N-terminal fgf variants having increased receptor selectivity and uses thereof

ABSTRACT

The present invention relates to the design, manufacture and use of fibroblast growth factor (FGF) polypeptides having improved receptor specificity. In particular, the invention relates to isolated FGF2 and FGF4 polypeptides that include a truncated N-terminus and optionally N-terminal amino acid substitutions. The present invention provides polypeptides, nucleic acids encoding the polypeptides, compositions of the same and methods for use thereof.

FIELD OF THE INVENTION

The present invention relates to fibroblast growth factor (FGF)variants, having improved receptor specificity. In particular, theinvention relates to isolated FGF2 and FGF4 polypeptides having specificN-terminus modifications including N-terminal truncations and N-terminalamino acid substitutions. The present invention provides polypeptides,nucleic acids encoding the polypeptides, compositions comprising sameand methods for use thereof.

BACKGROUND OF THE INVENTION Fibroblast Growth Factors and theirReceptors

Fibroblast growth factors (FGFs) comprise a large family ofevolutionarily conserved polypeptides involved in a variety ofbiological processes including morphogenesis, angiogenesis, and tissueremodeling as well as in the pathogenesis of numerous diseases. Thevarious members of this family stimulate the proliferation of a widespectrum of cells, including those deriving from mesenchymal,endothelial, epithelial and neuroectodermal origin. FGFs are expressedin a strict temporal and spatial pattern during development and haveimportant roles in patterning and limb formation (reviewed in Ornitz,2000). All members of the FGF family share a homology core domain ofabout 120 amino acids, 28 aa residues are highly conserved and four areidentical. The adjacent N- and C-termini are of variable length andshare limited homology. The core domain comprises both the primaryreceptor binding sites and a heparin-binding domain, which are distinctfrom each other (reviewed in Ornitz and Itoh, 2001).

Fibroblast growth factor 2, also known as FGF2, basic FGF, bFGF,prostatin and heparin binding growth factor 2, is highly conserved amongspecies and has been shown to stimulate the proliferation of a widevariety of cell types. Human FGF2 is expressed in several forms, a 210aa precursor, a 155 aa form, a 146 aa N-terminal truncated form andseveral others (reviewed in Okada-Ban et al., 2000). A method forpurifying recombinant FGF2 has been disclosed in WO 91/09126.

The biological response of cells to FGF is mediated through specific,high affinity (Kd 20-500 pM) cell surface receptors that possessintrinsic tyrosine kinase activity and are phosphorylated upon bindingof FGF. Five distinct Fibroblast Growth Factor Receptors (FGFRs) havebeen identified, FGFR1-4 are transmembrane-protein kinases while FGFR5lacks a tyrosine kinase domain. The FGFR extracellular domain consistsof three immunoglobulin-like (Ig-like) domains (D1, D2 and D3), aheparin binding domain and an acidic box. Alternative splicing of D3 inFGFR1-3 mRNAs generates six different receptor subtypes, each havingunique ligand specificity and tissue distribution pattern.

Another critically functional component in receptor activation is thebinding to soluble heparin or a heparan sulfate proteoglycan. Differentmodels have been proposed to explain the role of heparan sulfateproteoglycans (HSPG) in FGF signaling, including the formation of afunctional tertiary complex between FGF, FGFR and an HSPG (Yayon et al.,1991). Most naturally occurring heparan sulfate are incapable ofpromoting heparin dependent high affinity FGF receptor binding andactivation (Aviezer et al., 1994). Moreover, heparan sulfate which islocally secreted by cells, masks receptor specificity of the FGFligands.

FGFRs and Disease

A number of birth defects affecting the skeleton are associated withmutations in the genes encoding FGF receptors. Certain FGFRs have beenimplicated in certain malignancies and proliferative diseases. FGFR3 isthe most frequently mutated oncogene in transitional cell carcinoma(TCC) of the bladder where it is mutated in about 50% of the cases; theFGFR3IIIc isoform is ectopically expressed in 15-20% of patients withmultiple myeloma and is over expressed in the white blood cells ofchronic myeloid leukemia (CML) patients. A mutation in FGFR3 is linkedto cervical carcinoma. FGFR4 was shown to be associated with pituitarytumors and breast cancer progression. In contrast, certain FGF ligandshave been shown to be highly useful for treating indications includingwounds (U.S. Pat. Nos. 4,950,483, 5,859,208 and 6,294,359), myocardialinfarction (U.S. Pat. Nos. 4,296,100 and 4,378,347), skeletal disorders(U.S. Pat. Nos. 5,614,496 and 5,656,598) and for remodeling cardiactissue (U.S. Pat. No. 6,352,971).

FGF Variants and Receptor Specificity

All members of the FGF family share a homology core domain of about 120amino acids (aa), 28 aa residues are highly conserved and four areidentical. Structural studies on several FGFs identified twelveantiparallel β strands each one adjacent to (β-loops comprising the coreregion, conserved throughout the family. The core domain comprises theprimary FGFR and heparin binding sites. Receptor binding regions aredistinct from heparin binding regions (reviewed in Ornitz and Itoh,2001).

In view of the large number of FGFs and FGF receptor variants, a majorquestion regarding FGF function is their receptor specificity orselectivity. Most FGF ligands bind more than one receptor subtype andsuch a degree of cross-reactivity is shared between all FGF receptors,demonstrating a highly redundant signaling network. All FGFRs tested sofar bind FGF 1 (acidic FGF, aFGF) with moderate to high affinity,further demonstrating the apparent redundancy in the FGF system (Ornitzet al., 1996).

Various types of FGF variants are known in the art. U.S. Pat. No.6,294,359 discloses agonist and antagonist analogs of FGF2 that compriseamino acid substitutions in the C78 and C96 residues. U.S. Pat. No.5,352,589 discloses derivatives of FGF2 that act as antagonists orsuperagonists. One particular construct comprises a human or bovinederivative wherein amino acids 27-32 (KDPKRL) have been deleted. Wong etal., (1995) identified putative heparin binding domains in FGF1 (154 aaform) based on consensus sequence motifs, including amino acids 22-27(YKKPKL). Nevertheless, according to that citation substitution ofLys23, Lys24 or Lys26 with glycine residues had no effect on theactivity of FGF 1.

Attempts have been made to achieve altered FGF receptor specificity bymutating or truncating the ligands, by means of mutations introduced atcertain locations within the gene encoding for the proteins. Certaintruncated and mutated variants have been disclosed by some of theinventors of the present invention in PCT publications WO 02/36732 andWO 03/094835. International patent applications WO 02/36732 and WO03/094835 of some of the applicants of the present invention, discloseFGF variants having at least one amino acid substitution in the β8-β9loop, and/or an N- and/or C-terminal truncation, having increasedreceptor specificity to one receptor subtype compared to thecorresponding wild type FGF. PCT publication WO 02/36732 disclosesspecific FGF9 variants having 36, 44 or 63 amino acid truncations at theN-terminus. The shortest variant was shown to retain weak activitytowards FGFR3 while losing almost all activity towards FGFR1. PCTpublication WO 03/094835 teaches an FGF4 variant having both anN-terminal truncation (55 amino acids) and an amino acid substitution inthe β8-β9 loop, the variant exhibiting enhanced receptor specificitytowards FGFR3 with substantially unchanged activity towards FGFR1 andFGFR2.

Several investigators have demonstrated FGF mutants and variantsaffecting receptor and heparin binding. Kuroda et al., (1999)demonstrated that a full-length FGF4 polypeptide (191 aa) and anN-terminal truncated version containing 134 amino acid residues exhibitcomparable cellular proliferation on NIH-3T3 cells and increase of bonedensity. The shortest form of FGF4 tested, containing only 111 aminoacid residues, exhibited limited growth stimulatory activity.

U.S. Pat. No. 5,998,170 discloses a biologically active FGF16 moleculehaving from one to thirty-four amino acids deleted from the N-terminusor from one to eighteen amino acids deleted from the C-terminus. Thetruncated ligands were shown to retain biological activity includinghepatocellular proliferation and increased production of triglyceridesand serum proteins, when administered to animals.

X-ray crystallography has been used in an attempt to study the basis ofspecificity of FGFs to their receptors (Plotnikov et al., 2000; Olsen,et al., 2004; Mohammadi et al., 2005). The role of the N-terminal domainof the FGFs was resolved in only a few of the abovementioned crystalstructures. Olsen et al., (2004) compared receptor binding of a fulllength FGF1 (155 aa) to a N-terminal truncated form (21-155) and showthat the N-terminus of FGF1 may be relevant to binding and activation ofthe FGFR3c isoform. The (21-155) form also exhibits reduced FGFR2 andFGFR3 phosphorylation.

Plotnikov et al., (2000) determined the crystal structures of FGF1 andFGF2 complexed with the ligand binding domains (Ig-like domains 2 and 3)of FGFR1 and FGFR2, respectively and shows that certain N-terminalresidues of FGF, in particular Phe 17 and Lys18 of FGF2 (Lys27 of 155 aaform), could be in contact with the D3 domain of FGFR2. The authorsspeculated, but did not provide experimental evidence, that amino acids7-13 (NYKKPKL) of FGF1 play a role in receptor binding. The deletion ofthat specific sequence, which had originally been proposed to be anuclear localization sequence, reduces the ability of FGF1 to inducecell proliferation in endothelial cell lines by about 250-fold (Imamuraet al., 1990). These findings neither suggest nor teach that thedeletion of that specific sequence would affect receptor selectivity.

Seno et al., (1990) teach certain bFGF variants having N- and C-terminustruncations and have characterized their ability to bind heparin. Themitogenic activity of those variants in BALB/c3T3 cells was determinedand the N14 variant (corresponding to a 22 amino acid truncation of the155 aa bFGF species) showed an activity of 68% that of the mature formof bFGF. A much larger truncation, N41, which corresponds to a 49 aminoacid truncation of the 155 aa species, exhibits only about 2% mitogenicactivity. There is neither teaching nor suggestion of a truncated FGFexhibiting receptor selectivity toward one or another FGFR species.

In another study the basic residues in an analogous stretch of FGF2 (aa27-31, KDPKR, 155 aa form) were modified to neutral glutamine residues,specifically K27Q, K30Q and R31Q (Presta et al., 1993). That mutantretains receptor binding capacity and mitogenic activity on endothelialcells yet exerts reduced uPA inducing activity.

The above disclosures show that modifications in certain N-terminalresidues affect cell proliferation, yet they neither teach nor suggestthat mutations or substitutions in N-terminal residues of FGF wouldaffect receptor selectivity.

A hexapeptide, consisting of N-terminal amino acids 13-18 of FGF2 (146aa form, corresponding to aa 22-27 of the 155 aa form), was shown toinhibit binding of FGF2 to FGFR-1, implicating this motif in receptorbinding (Yayon et al, 1993). There was neither teaching nor suggestionof receptor specificity.

Attempts have been made to alter FGF receptor specificity and heparinbinding by means of site directed mutagenesis within the FGF genes. U.S.Pat. No. 5,512,460 discloses a biologically active FGF9 (glia activatingfactor, GAF) molecule comprising N-terminus and C-terminus truncationsof 53 aa and 13 aa, respectively. U.S. Pat. No. 5,571,895 discloses anN-terminus 54 aa deletion yielding a 154 aa protein retaining itsbiological activity, as measured by glial cell growth activity.

U.S. Pat. No. 5,491,220 to one of the inventors of the presentapplication discloses structural analogues of FGF2 comprisingsubstitution of the β9-β10 loop having altered biological properties andbinding specificity.

Springer et al., (1994) identified two FGFR binding sites on FGF2, thefirst includes hydrophobic residues Y24, Y103, L140 and M142 and polarresidues R44, and N101. The author concludes that R44 and N101 were theonly polar residues observed to be important in the primary bindinginteraction between FGF2 and FGFR.

In an attempt to identify FGF2 antagonists having reduced bindingaffinity towards FGFR1. Zhu et al. (1997) tested N101A, N102A, Y103A,N104A and T105A muteins and their binding affinity to FGFR1. Thesecorrespond to amino acid residues N110, N111, Y112, N113 and T114 of the155 aa species. The N101A and N102A muteins were shown to have FGFR1binding similar to that of the wild type protein, while the N104A muteinexhibited 400 fold reduced FGFR1 binding.

There is neither teaching nor suggestion of receptor selectivity in theabove-cited references.

Bellosta et al., (2001) have disclosed mutated and truncated FGF4variants having reduced receptor binding and a low mitogenic potential.Of interest is a truncated variant, which lacks 78 N-terminal aminoacids and, according to the published data, retains the core domain,exhibiting FGFR binding affinities similar to that of the wild typeligand. Certain mutations within the core domain were shown to have adeleterious effect on both DNA synthesis and receptor binding.

The extensive efforts made to produce truncation, deletion and pointmutation variants in FGF have resulted in certain alterations inreceptor specificity. There remains an unmet need for highly active andselective ligands for the various FGF receptor isoforms, useful inselective stimulation or inhibition of these receptors, therebyaddressing the clinical manifestations associated with receptormutations, and modulation of various biological functions.

It is to be understood that known variants of FGF are excludedexplicitly from the present invention.

A need for FGF variants having increased receptor selectivity ismanifest. Lack of receptor selectivity is often detrimental to tissuerepair and regeneration both ex vivo and in vivo. For instance, FGFR1activation is often critical for cell survival and proliferation; hence,a ligand having enhanced FGFR1 specificity would be ideal for supportingphysiological FGF mediated activities in processes such as woundhealing, independent of the heparan sulfate environment.

SUMMARY OF THE INVENTION

The present invention provides Fibroblast Growth Factor (FGF)polypeptides that show an increase in receptor selectivity when comparedto the corresponding wild type protein. In particular, the inventionprovides FGF polypeptides in which the wild type amino acid sequence hasbeen modified to introduce an N-terminal truncation. It is now disclosedthat specific N-terminal modified FGF2 and FGF4 polypeptide variantshaving increased receptor selectivity, are useful in research andclinical applications.

The present inventors have found unexpectedly that certain N-terminalvariants of FGF2 and FGF4 have enhanced receptor selectivity. Wild typeFGF2 is known to activate FGFR1, FGFR2 and FGFR3 indiscriminately. Incontrast, certain FGF2 variants of the present invention are now shownto selectively activate only one or two of those receptors. The clinicalapplications are manifold, including receptor selective agonist ligandsuseful for, inter alia, wound healing, bone and cartilage regenerationand stem cell proliferation and differentiation.

In one aspect the present invention provides an isolated N-terminalmodified fibroblast growth factor polypeptide selected from FGF2 andFGF4. Accordingly, the present invention provides an isolated variantpolypeptide of a fibroblast growth factor selected from the groupconsisting of FGF2 and FGF4 comprising an N-terminal deletion; whereinthe FGF2 variant polypeptide retains between 0 and 11 amino acidresidues at the N-terminus extending beyond the Leu-Tyr-Cys (LYC) motifof the β1 strand of the core domain; and wherein the variant polypeptidehas increased receptor selectivity when compared to the correspondingisolated wild type FGF polypeptide by a gain of activity or loss ofactivity by at least a factor of two toward at least one receptorsubtype but not toward all FGFR subtypes.

The FGF2 core domain is set forth in SEQ ID NO:1; the FGF4 core domainis set forth in SEQ ID NO:2. For reference purposes, the 155 aa FGF2polypeptide (18 kD isoform) is set forth in SEQ ID NO:3 (NP_(—)001997)and the 206 aa FGF4 polypeptide (FGF4 precursor) is set forth in SEQ IDNO:4 (NP_(—)001998.).

The amino acid sequences of the FGF2 and FGF4 core domains are shown inFIG. 1A. The amino acid sequences of the β1 strand are bolded. TheLeu-Tyr-Cys (LYC) motif is underlined.

In some embodiments the isolated FGF variant polypeptide is an FGF2variant. In certain embodiments the FGF2 retains between 0 and 5 aminoacid residues at the N-terminus extending beyond the LYC motif of the β1strand of the core domain. In some embodiments the variant polypeptideis selected from a group of polypeptides having an amino acid sequenceset forth in any one of SEQ ID NOS: 7-12. In one embodiment the FGF2variant polypeptide has a sequence set forth in SEQ ID NO:7. In anotherembodiment the FGF2 variant polypeptide has a sequence set forth in SEQID NO:12.

A description of the FGF2 N-terminal variant polypeptides is set forthhereinbelow. It is to be noted that the N-terminal methionine residue(Met) is required for expression in the bacterial expression system, yetit is generally post-translationally cleaved and a polypeptide lackingthe first Met is obtained. However, it is to be understood that variantpolypeptides having the N-terminal methionine are encompassed in thepresent application.

The FGF2 variant polypeptides are characterized as follows:

SEQ ID NO:5, represents FGF2^(Δ24), having a 23 amino acid N-terminaltruncation with the Gly24 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:34. Theexpressed FGF2^(Δ24) polypeptide has a 7 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:6, represents FGF2^(Δ25), having a 24 amino acid N-terminaltruncation with the His25 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:35. Theexpressed FGF2^(Δ25) polypeptide has a 6 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:7, represents FGF2^(Δ26), having a 25 amino acid N-terminaltruncation with the Phe26 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:36. Theexpressed FGF2^(Δ26) polypeptide has a 5 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:8, represents FGF2^(Δ27), having a 26 amino acid N-terminaltruncation with the Lys27 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:37. Theexpressed FGF2^(Δ27) polypeptide has a 4 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:9, represents FGF2^(Δ28), having a 27 amino acid N-terminaltruncation with the Asp28 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:38. Theexpressed FGF2^(Δ28) polypeptide has a 3 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:10, represents FGF2^(Δ29), having a 28 amino acid N-terminaltruncation with the Pro29 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:39. Theexpressed FGF2^(Δ29) polypeptide has a 2 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:11, represents FGF2^(Δ30), having a 29 amino acid N-terminaltruncation with the Lys30 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:40. Theexpressed FGF2^(Δ30) polypeptide has a 1 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:12, represents FGF2^(Δ31), having a 30 amino acid N-terminaltruncation with the Arg31 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:41. Theexpressed FGF2^(Δ31) polypeptide has no N-terminal sequence precedingthe LYC motif of the β1 strand of the core domain. The sequence mayfurther comprise a Met residue at the N-terminus.

In some embodiments the FGF variant polypeptide further comprises atleast one additional modification in its polypeptide sequence, whereinthe modification is selected from an amino acid deletion, an amino acidsubstitution and an amino acid insertion. In some embodiments theadditional modification is an amino acid residue substitution in thebeta8-beta9 loop. In one embodiment the variant polypeptide is an FGF2variant polypeptide denoted herein FGF2^(Δ26)N111G having sequence setforth in SEQ ID NO:13. The corresponding polynucleotide sequence is setforth in SEQ ID NO:42. The sequence may further comprise a Met residueat the N-terminus.

For convenience, the above-mentioned FGF2 variants are listed in table Iwith their sequence identifiers.

TABLE I FGF2 variants and their associated sequence identifiers. SEQ IDNO: SEQ ID NO: POLYPEPTIDE POLYNUCLEOTIDE FGF2^(Δ24) 5 34 FGF2^(Δ25) 635 FGF2^(Δ26) 7 36 FGF2^(Δ27) 8 37 FGF2^(Δ28) 9 38 FGF2^(Δ29) 10 39FGF2^(Δ30) 11 40 FGF2^(Δ31) 12 41 FGF2^(Δ26)N111G 13 42

In other embodiments the variant polypeptide comprises at least oneamino acid substitution in the retained N-terminus sequence. In someembodiments the variant polypeptide is an FGF2 variant having a sequenceset forth in any one of SEQ ID NO:14-16.

SEQ ID NO:14 denoted herein FGF2^(Δ24)H25X1, wherein X1 is an amino acidresidue other than His. In some embodiments X1 is selected from thegroup of amino acid residues consisting of Q, K and V. In certainembodiments X1 is selected from K and V. The sequence may furthercomprise a Met residue at the N-terminus.

SEQ ID NO:15 denoted herein FGF2^(Δ24) F26X2, wherein X2 is an aminoacid residue other than Phe. In some embodiments X2 is selected from thegroup of amino acid residues consisting of D, Q, and K. The sequence mayfurther comprise a Met residue at the N-terminus.

SEQ ID NO:16 denoted herein FGF2^(Δ24)H25X3-F26X4, wherein X3 is anamino acid residue other than His and X4 is an amino acid residue otherthan Phe. In some embodiments X3 is selected from the group of aminoacid residues consisting of D, Q, K and V and X4 is selected from thegroup of amino acid residues consisting of D, Q and K. The sequence mayfurther comprise a Met residue at the N-terminus.

In some embodiments the additional modification is an amino acid residuesubstitution at the N-terminus. In one embodiment the variantpolypeptide is an FGF2 variant polypeptide in which Phe 26 is replacedby Gln denoted herein FGF2^(F26Q) having the sequence set forth in SEQID NO:90. The corresponding polynucleotide sequence is set forth in SEQID NO:91.

According to some embodiments the FGF2 variant is selected from any oneof SEQ ID NO:17-23 or 25-33, as characterized hereinbelow in Table II:

TABLE II FGF2 variants and their associated sequence identifiers.POLYPEPTIDE POLYNUCLEOTIDE SEQ ID NO: SEQ ID NO: Δ24H25D 17 43 Δ24H25Q18 44 Δ24H25V 19 45 Δ24H25K 20 46 Δ24F26D 21 47 Δ24F26Q 22 48 Δ24F26K 2349 Δ24F26Y 24 50 FGF2^(Δ24)H25D-F26D 25 51 FGF2^(Δ24)H25D-F26Q 26 52FGF2^(Δ24)H25D-F26K 27 53 FGF2^(Δ24)H25Q-F26D 28 54 FGF2^(Δ24)H25Q-F26K29 55 FGF2^(Δ24)H25V-F26D 30 56 FGF2^(Δ24)H25V-F26Q 31 57FGF2^(Δ24)H25V-F26K 32 58 FGF2^(Δ24)H25K-F26Q 33 59

In some embodiments the isolated FGF variant polypeptide is an FGF4variant. In certain embodiment the isolated FGF4 variant polypeptideretains from between 8 to 11 amino acid residues at the N-terminusextending beyond the LYC motif of the β1 strand of the core domain. TheFGF4 variant polypeptides may further comprise a Met residue at theN-terminus.

In some embodiments the N-terminal truncated polypeptide is an FGF4variant polypeptide selected from the group set forth in any one of SEQID NO:63-65. Variant sequences SEQ ID NO:60-62 have a similar activityto that of the FGF4 wild type, SEQ ID NO:67-68 are provided forcomparison.

SEQ ID NO:60, representing FGF4^(Δ72), has a 71 amino acid N-terminaltruncation with the Gly72 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:69. Theexpressed FGF4^(Δ72) polypeptide has a 13 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:61, representing FGF4^(Δ73), has a 72 amino acid N-terminaltruncation with the Ala73 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:70. Theexpressed FGF4^(Δ73) polypeptide has a 12 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:62, representing FGF4^(Δ74), has a 73 amino acid N-terminaltruncation with the Gly74 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:71. Theexpressed FGF4^(Δ74) polypeptide has an 11 amino acid N-terminalsequence preceding the LYC motif of the β1 strand of the core domain.The sequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:63, representing FGF4^(Δ75), has a 74 amino acid N-terminaltruncation with the Asp75 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:72. Theexpressed FGF4^(Δ72) polypeptide has a 10 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:64, representing FGF4^(Δ76), has a 75 amino acid N-terminaltruncation with the Tyr76 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:73. Theexpressed FGF4^(Δ76) polypeptide has a 9 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:65, representing FGF4^(Δ77), has a 76 amino acid N-terminaltruncation with the Leu77 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:74. Theexpressed FGF4^(Δ77) polypeptide has an 8 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:66 representing FGF4^(Δ78), has a 77 amino acid N-terminaltruncation with the Leu78 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:75. Theexpressed FGF4^(Δ78) polypeptide has a 7 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus. Thetruncated FGF4^(Δ78) polypeptide sequence has been disclosed in Bellostaet al., (2001) but according to the published data, exhibited bindingaffinities similar to that of the wild type ligand.

SEQ ID NO:67 representing FGF4^(Δ79), has a 78 amino acid N-terminaltruncation with the Gly79 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:76. Theexpressed FGF4^(Δ79) polypeptide has a 6 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

SEQ ID NO:68 representing FGF4^(Δ80), has a 79 amino acid N-terminaltruncation with the Ile80 replaced by a Met residue. This polypeptide isencoded by a polynucleotide sequence set forth in SEQ ID NO:77. Theexpressed FGF4^(Δ78) polypeptide has a 5 amino acid N-terminal sequencepreceding the LYC motif of the β1 strand of the core domain. Thesequence may further comprise a Met residue at the N-terminus.

The sequence identifier numbers can be found hereinbelow in Table III.

TABLE III FGF4 variant and their associated sequence identifiers. SEQ IDNO: SEQ ID NO: POLYPEPTIDE POLYNUCLEOTIDE FGF4^(Δ72) 60 69 FGF4^(Δ73) 6170 FGF4^(Δ74) 62 71 FGF4^(Δ75) 63 72 FGF4^(Δ76) 64 73 FGF4^(Δ77) 65 74FGF4^(Δ78) 66 75 FGF4^(Δ79) 67 76 FGF4^(Δ80) 68 77

According to certain embodiments the N-terminal truncated FGF is an FGF4polypeptide variant having amino acid sequence selected from any one ofSEQ ID NO:63-65. According to another aspect the present inventionprovides a polynucleotide molecule whose sequence encodes an N-terminalFGF4 variant polypeptide of the invention, the polynucleotide sequenceset forth in any one of SEQ ID NOS:72-74.

In a second aspect the present invention provides an isolatedpolynucleotide sequence encoding a variant polypeptide of a fibroblastgrowth factor selected from the group consisting of FGF2 and FGF4comprising an N-terminal deletion; wherein the variant polypeptideretains between 0 and 11 amino acid residues at the N-terminus extendingbeyond the LYC motif of the β1 strand of the core domain; and whereinthe variant polypeptide has increased receptor selectivity when comparedto the corresponding wild type FGF polypeptide by a gain of activity orloss of activity by at least a factor of two toward at least onereceptor subtype but not toward all FGFR subtypes.

In one aspect the present invention provides a vector comprising thepolynucleotide sequences set forth in any one of SEQ ID NOS:34-59 or SEQID NOS:69-74 or SEQ ID NOS:76-77. According to some embodiments thepresent invention provides a vector comprising any one of polynucleotidesequences set forth in any one of SEQ ID NOS:36-42, SEQ ID NOS:43-49,SEQ ID NOS:51-59, SEQ ID NOS:72-74.

In another aspect, the present invention provides a host cell comprisinga vector, the vector comprising a sequence set forth in any one of SEQID NOS:34-42 or SEQ ID NOS:43-59 or SEQ ID NOS:69-74 or SEQ IDNOS:76-77. According to some embodiments the present invention providesa host cell comprising a vector, the vector comprising any one ofpolynucleotide sequences set forth in any one of SEQ ID NOS:36-42, SEQID NOS:43-49, SEQ ID NOS:51-59 or SEQ ID NOS:72-74.

In another aspect the present invention provides a pharmaceuticalcomposition comprising as an active ingredient an isolated N-terminalmodified fibroblast growth factor polypeptide selected from FGF2 andFGF4. Accordingly the present invention provides an isolated variantpolypeptide of a fibroblast growth factor selected from the groupconsisting of FGF2 and FGF4 comprising an N-terminal deletion; whereinthe variant polypeptide retains between 0 and 11 amino acid residues atthe N-terminus extending beyond the LYC motif of the β1 strand of thecore domain; and wherein the variant polypeptide has increased receptorselectivity when compared to the corresponding isolated wild type FGFpolypeptide by a gain of activity or loss of activity by at least afactor of two toward at least one receptor subtype but not toward allFGFR subtypes; and a pharmaceutically acceptable diluent or carrier.

According to another aspect the present invention provides apharmaceutical composition comprising a therapeutic amount of at leastone isolated polynucleotide sequence encoding an isolated variant FGF2polypeptide comprising an N-terminal deletion; wherein the variantpolypeptide retains between 0 and 5 amino acid residues at theN-terminus extending beyond the LYC motif of the β1 strand of the coredomain; and wherein the variant polypeptide has increased receptorselectivity when compared to the corresponding isolated wild type FGF2polypeptide by a gain of activity or loss of activity by at least afactor of two toward at least one receptor subtype but not toward allFGFR subtypes, and a pharmaceutically acceptable diluent or carrier.According to one embodiment the pharmaceutical composition comprises atleast one polypeptide sequence having an amino acid sequence set forthin any one of SEQ ID NO:7-12, SEQ ID NO:17-23 or SEQ ID NO:25-33; and apharmaceutically acceptable carrier, diluent or excipient. In someembodiments the pharmaceutical composition comprises a polypeptidehaving amino acid sequence set forth in SEQ ID NO:7.

According to another aspect the present invention provides apharmaceutical composition comprising a therapeutic amount of anisolated polynucleotide sequence encoding an isolated variant FGF4polypeptide comprising an N-terminal deletion; wherein the variantpolypeptide retains between 8 and 11 amino acid residues at theN-terminus extending beyond the LYC motif of the β1 strand of the coredomain; and wherein the variant polypeptide has increased receptorselectivity when compared to the corresponding isolated wild type FGF4polypeptide by a gain of activity or loss of activity by at least afactor of two toward at least one receptor subtype but not toward allFGFR subtypes, and a pharmaceutically acceptable diluent or carrier.According to one embodiment the pharmaceutical composition comprises atleast one polypeptide sequence having an amino acid sequence set forthin any one of SEQ ID NO:63-65; and a pharmaceutically acceptablecarrier, diluent or excipient.

According to another aspect a single point mutation at the N-terminushas been found advantageous in terms of receptor selectivity, evenwithout N-terminal truncation. According to another embodiment the FGFvariant is an FGF2 polypeptide variant having amino acid sequence setforth in SEQ ID NO:90. In a specific embodiment the present inventionprovides an isolated polynucleotide sequence set forth in SEQ ID NO:91.In a specific embodiment the present invention provides a vectorcomprising the polynucleotide sequence set forth in SEQ ID NO:91. Inanother embodiment the present invention provides a pharmaceuticalcomposition comprising as an active ingredient the isolated N-terminalmodified fibroblast growth factor polypeptide set forth in SEQ ID NO:90.According to another embodiment the present invention provides apharmaceutical composition comprising a therapeutic amount of theisolated polynucleotide sequence set forth in SEQ ID NO:91.

In some embodiments the pharmaceutical composition of the presentinvention is formulated for administration via intra-articular,intravenous, intramuscular, subcutaneous, intradermal, or intrathecalroutes. In certain embodiments the pharmaceutical composition isformulated for administration to the site of a bone fracture orcartilage lesion. In other embodiment the pharmaceutical composition isformulated for application to a wound.

In another aspect the present invention provides a method of treating anindividual in need thereof comprising the step of administering to thatindividual a pharmaceutical composition according to the presentinvention. The present invention includes methods of treating a subjectwith a wound, a bone disorder, a cartilage disorder. In otherembodiments the present invention provides a method of treating asubject with coronary and peripheral vascular disease. These methods oftreatment the abovementioned disorders or diseases comprise theadministration of any of the following sequences set forth in SEQ IDNOS: 7-13, SEQ ID NOS: 17-23, SEQ ID NOS: 25-33, SEQ ID NOS:63-65, andtheir corresponding nucleotides in SEQ ID NOS: 36-42, SEQ ID NOS: 43-49,SEQ ID NOS: 51-59 and SEQ ID NOS: 72-74.

In another embodiment the present invention provides a method oftreating a subject with a wound, a bone disorder, a cartilage disorder,a coronary and peripheral vascular disease with the administration of apolypeptide having an amino acid sequence set forth in SEQ ID NO:66, orits corresponding polynucleotide having a sequence set forth in SEQ IDNO:75.

In yet another aspect the present invention provides a method ofinducing cellular expansion, comprising the steps of:

a. isolating a population of cells to be expanded; and

b. exposing said cells to an N-terminal FGF polypeptide variantaccording to the present invention.

In some embodiments the population of cells to be expanded compriseshematopoietic cells. In other embodiments the population of cells to beexpanded the cells are selected from stem cells or progenitor cells.Cells suitable for proliferation include cells selected fromchondrocytes, osteoblasts, hepatocytes, fibroblasts or mesenchymal,endothelial, epithelial, urothelial, endocrine, neuronal, pancreatic,renal and ocular cell types.

In yet another aspect the present invention provides methods for the useof an isolated FGF polypeptide of the present invention to preparemedicaments useful for treating various diseases and disorders. In oneembodiment the present invention provides the use of isolated FGFpolypeptides of the present invention to prepare medicaments useful inbone and cartilage formation and regeneration, wound healing,neovascularization and treating FGFR related skeletal and proliferativedisorders.

The isolated polypeptide variants of the present invention are usefulfor a variety of therapeutic applications including wound healing,induction of angiogenesis, tissue repair and tissue regeneration. Itwill be appreciated that the therapeutic methods of the invention thesubject to be treated is preferably a mammal, more preferably a human.

The abbreviations used herein correspond to the one letter amino acidcode followed by the number designating the amino acid position in the155 aa form of FGF2 (SEQ ID NO:3), the 206 aa form of FGF4 (SEQ IDNO:4), and the one letter amino acid code for the substituted aminoacid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. The amino acid residues comprising the core domains of humanFGF2 (SEQ ID NO:1) and human FGF4 (SEQ ID NO:2). FIG. 1B. Human FGF2polypeptide sequence and N-terminal variants. Arrows indicate thepenultimate amino acid of FGF2^(Δ24), FGF2^(Δ25), FGF2^(Δ26) andFGF2^(Δ31). FIG. 1C. Human FGF4 polypeptide sequence and N-terminalvariants. An arrow indicates the penultimate amino acid of FGF4^(Δ78).

FIG. 2. Structure function analysis of FGF4. Successive N-terminaldeletion mutants were added to FDCP-FGFR1 (white columns) or FDCP-FGFR3(black columns) cells. Cell proliferation was determined 2 days followedby the XTT assay.

FIGS. 3A-3C. Activity of N-terminal truncated FGF2 (FGF2^(Δ26)) towardsdifferent FGF receptors. FGF2^(Δ26) (squares) retains wild-type activitytowards FGFR1 (FIG. 3A), while exhibiting reduced activity towards FGFR2(FIG. 3B) and FGFR3 (FIG. 3C) in comparison to wild-type (diamonds)activity. FDCP cells were incubated 2 days with the wild-type FGF2 orFGF2^(Δ26) ligands and cell proliferation was measured by XTT.

FIG. 4. RCJ cells expressing one of FGFR1, FGFR2 or FGFR3 were incubatedfor 5 minutes with FGF2 or FGF2^(Δ26). Whole cell lysates were thenanalyzed by Western blot analysis using an anti-phosphorylated ERK(MAPK) antibody.

FIGS. 5A-5B. FGFR1 selectivity of N-terminal truncated FGF2 variants inFDCP cells. FDCP cells expressing FGFR1 (FIG. 5A) or FGFR3 (FIG. 5B)were stimulated with increasing amount of wild-type (FGF2; diamonds),FGF2^(Δ26) (squares) or FGF2^(Δ31) (triangles). The resulting cellproliferation was measured by XTT.

FIGS. 6A-6C. The activity of the N-terminal truncated variantsFGF2^(Δ24) (small squares), FGF2^(Δ25) (triangles) and FGF2^(Δ26) (largesquares) compared to activity of wild-type FGF2 (diamonds) on FGFR1(FIG. 6A), FGFR3 (FIG. 6B) and FGFR4 (FIG. 6C). Cell proliferation atthe indicated dose of each ligand was measured by XTT analysis.

FIGS. 7A-7N. Activation of FGFR1 by N-terminal truncated FGF2 variantshaving substitutions at amino acid residues 25 and 26. FDCP-FGFR1 cellswere cultured with increasing levels of FGF2 mutants for 2 days. Then,cell proliferation was measured by XTT analysis. Wild-type FGF2(squares), FGF2^(Δ24) (denoted D24; diamonds), FGF2^(Δ25) (denoted D25;rectangles) and FGF2^(Δ26) (denoted D26; circles) were included in theassay as controls. The following variants (triangles) are compared tothe controls at each figure in the following manner: FGF2^(Δ24H25D)(FIG. 7A), FGF2^(Δ24H25Q) (FIG. 7B), FGF2^(Δ24H25V) (FIG. 7C),FGF2^(Δ24F26D) (FIG. 7D), FGF2^(24F26Q) (FIG. 7E), FGF2^(Δ24F26K) (FIG.7F), FGF2^(Δ24H25DF26D) (FIG. 7G), FGF2^(Δ24H25DF26Q) (FIG. 7H),FGF2^(Δ24H25DF26K) (FIG. 7I), FGF2^(Δ24H25QF26D) (FIG. 7J),FGF2^(Δ24H25QF26K) (FIG. 7K), FGF2^(Δ24H25VF6D) (FIG. 7L),FGF2^(Δ24H25VF26Q) (FIG. 7M), FGF2^(Δ24H25VF26K) (FIG. 7N). The data areaverage of duplicate cultures.

FIGS. 8A-8N. Activation of FGFR2 by N-terminal truncated FGF2 variantshaving substitutions at residues 25 and 26. FDCP-FGFR2 cells werecultured with increasing levels of FGF2 mutants for 2 days. Then, cellproliferation was measured by XTT analysis. Wild-type FGF2 (squares),FGF2^(Δ24) (diamonds), FGF2^(Δ25) (rectangles) and FGF2^(Δ26) (circles)were included in the assay as controls. The following variants(triangles) are compared to the controls at each figure in the followingmanner: FGF2^(Δ24H25D) (FIG. 8A), FGF2^(Δ24H25Q) (FIG. 8B),FGF2^(Δ24H25V) (FIG. 8C), FGF2^(Δ24F26D) (FIG. 8D), FGF2^(Δ24F26Q) (FIG.8E), FGF2^(Δ24F26K) (FIG. 8F), FGF2^(Δ24H25DF26D) (FIG. 8G),FGF2^(Δ24H25DF26Q) (FIG. 8H), FGF2^(Δ24H25DF26K) (FIG. 8I),FGF2^(Δ24H25QF26D) (FIG. 8J), FGF2^(Δ24H25QF26K) (FIG. 8K),FGF2^(Δ24H25VF26D) (FIG. 8L), FGF2^(Δ24H25VF26Q) (FIG. 8M),FGF2^(Δ24H25VF26K) (FIG. 8N). The data are average of duplicatecultures.

FIGS. 9A-9J. Activation of FGFR3 by N-terminal truncated FGF2 variantshaving substitutions at residues 25 and 26. FDCP-FGFR3 cells werecultured with increasing levels of FGF2 variants for 2 days. Then, cellproliferation was measured by XTT analysis. As controls, wild-type FGF2,FGF2^(Δ24), FGF2^(Δ25) and FGF2^(Δ26) were included in the assay. Thefollowing variants are compared to the FGF2 wild-type (squares) in thefollowing manner: FGF2^(Δ24H25D) (diamonds), FGF2^(Δ24H25Q) (triangles)and FGF2^(Δ24H25V) (circles) in FIG. 9A; FGF2^(Δ24F26D)(triangles) inFIG. 9B; FGF2^(Δ24F26Q) (diamonds) and FGF2^(Δ24F26K) (circles) in FIG.9C; FGF2^(Δ24H25DF26D) (triangles), FGF2^(Δ24) (diamonds), FGF2^(Δ25)(rectangles) and FGF2^(Δ26) (circles) in FIG. 9D; FGF2^(Δ24H25DF26Q)(diamonds) and FGF2 (circles) in FIG. 9E; FGF2^(Δ24H25QF26D) (circles)in FIG. 9F; FGF2^(Δ24H25QF26K) (triangles) in FIG. 9G;FGF2^(Δ24H25VF26D) (triangles) in FIG. 9H; FGF2^(Δ24H25VF26Q) (circles)in FIG. 9I; and FGF2^(Δ24H25VF26K) (triangles) in FIG. 9J. The data areaverage of duplicate cultures.

FIGS. 10A-10J. Activation of FGFR4 by N-terminal truncated FGF2 variantshaving substitutions at residues 25 and 26. FDCP-FGFR4 cells werecultured with increasing levels of FGF2 variants for 2 days. Then, cellproliferation was measured by XTT analysis. As controls, wild-type FGF2,FGF2^(Δ24), FGF2^(Δ25) and FGF2^(Δ26) were included in the assay. It isnoteworthy that all substitutions which appear in the figure are inaddition to a Δ24 N-terminal truncation. The data are average ofduplicate cultures.

FIGS. 11A-11D. Heparin dependent activation of FGFR1 by N-terminaltruncated FGF2 variants having substitutions at residues 25 and 26.FDCP-FGFR1 cells were incubated with 1.5 ng/ml of the indicated FGF2mutants at limiting heparin levels and cell proliferation was measuredby XTT analysis. It is noteworthy that all substitutions which appear inthe figure are in addition to a Δ24 N-terminal truncation.

FIGS. 12A-12B. Activation of FGFR1 (FIG. 12A) and FGFR3 (FIG. 12B) byFGF2 N-terminal truncated variants at residues 25 and/or 26. N-terminaltruncated FGF2 with the indicated replacement at position 25 and/or 26were added at increasing concentrations to FDCP-FGFR1 or FDCP-FGFR3cells. The variants are denoted as follows: FGF2^(Δ24) (small squares),FGF2^(Δ24H25K) (diamonds), FGF2^(Δ24F26Q) (triangles), FGF2^(Δ24H25D)(large squares), FGF2^(Δ24H25DF26Q)(rectangles), and FGF2^(Δ24H25JF26Q)(circles). Cell proliferation (average duplicate cultures) was measured2 days later by XTT analysis.

FIGS. 13A-13F. Activity of FGF2^(F26Q) under saturating (FIGS. 13A-13C),or limiting (FIGS. 13D-13F) heparin concentrations. FDCP-FGFR1 (FIGS.13A, 13D), FDCP-FGFR2 (FIGS. 13B, 13E) or FDCP-FGFR3 (FIGS. 13C, 13F)cells were cultured with increasing FGF2 (squares) or FGF2^(F26Q)(diamonds) levels under saturating (5 μg/ml) or limiting (20 ng/ml)heparin concentrations. Cell proliferation was measured after 2 days byXTT. Data are average of 2 cultures +/−SD.

FIGS. 14A-14C. Activity of FGF2^(Δ24F26Y) on FDCP-FGFR1 (FIG. 14A),FDCP-FGFR2 (FIG. 14B) and FDCP-FGFR3 (FIG. 14C). FGF2^(Δ24F26Y)(circles), FGF2^(Δ24F26Q) (squares) or FGF2^(Δ24) (diamonds) were addedat increasing amounts to FDCP-FGFR1, FDCP-FGFR2 and FDCP-FGFR3 cells.Cell proliferation was evaluated 2 days later by XTT analysis.

FIG. 15. Effect of N-terminal truncated FGF2 variants on primary humanarticular chondrocytes proliferation. Primary human articularchondrocytes were isolated from cartilage piece and expanded for 10 daysin DMEM/F12, 20% human serum and 10 ng/ml of FGF2^(Δ26) orFGF2^(Δ31)+heparin(5 μg/ml). Every 3 days medium was replaced. Cellswere counted after 10 days.

FIGS. 16A-16B. Effect of N-terminal truncated FGF2 and FGF4 variants onhuman bone marrow derived mesenchymal stem cells (hBM-MSC)proliferation. Samples of human bone marrow were seeded on 10 cm platesin DMEM-LG, 20% HS, and 5 μg/ml LMW Heparin andFGF4^(Δ78)/FGF2^(Δ24F26Q)/no ligand (FIG. 16A) orFGF2^(Δ26)/FGF2^(Δ24F26Q)/no ligand. After 6 days the cells were usedfor proliferation assay: 1−5×10⁴ cells were seeded in 24 wells plate andin several time points cells were counted and reseeded.

FIG. 17. Histology analysis of pellet cultures produced from hBM-MSC.Human BM-MSCs were expanded in DMEM-LG medium supplemented with 20%human serum and 10 ng/ml FGF2, or FGF2^(Δ26)+Heparin (5 μg/ml). Pelletculture assay was performed on the cells expanded for 10-14 days. Thepellet cultures were incubated in differentiation medium for 21 days andhistologically analyzed. Histology included hematoxylin eosin (H&E),Alcian blue (AB), and Safranin O (SO) and immunohistochemistry withanti-Collagen II antibodies (Col II).

FIG. 18. Histology analysis of pellet cultures produced from hBM-MSC.Human BM-MSCs were expanded in DMEM-LG medium supplemented with 20%human serum and 10 ng/ml FGF2^(Δ26), or FGF2^(Δ24), orFGF2^(Δ31)+Heparin (5 μg/ml). Pellet culture assay was performed on thecells expanded for 14 days. The pellet cultures were incubated indifferentiation medium for 21 days and histologically analyzed.Histology included hematoxylin eosin (H&E), Alcian blue (AB), andSafranin O (SO) and immunohistochemistry with anti-Collagen IIantibodies (Col II).

FIGS. 19A-19B. qPCR analysis of Col II in pellet culture produced fromhBM-MSC expanded with FGF2, FGF2^(Δ26) variant and with no ligand forcomparison (FIG. 19A), or with FGF2^(Δ26), FGF2^(Δ31) and FGF2^(Δ24F26Q)variants and with no ligand for comparison (FIG. 19B). Expression of ColII was analyzed by qPCR of RNA isolated from pellet culture producedfrom hBM-MSC expanded for 14 days.

FIG. 20. In-vitro osteogenesis assay of hBM-MSC expanded in the presenceof different ligands. Mesenchymal stem cells (MSC) isolated from humanbone marrow sample were expanded for 14 days in MSC expansion mediumcontaining 10 ng/ml FGF2^(Δ24F26Q) or FGF4^(Δ78) and 5 μg/ml heparin.The cells (1×10⁵ per well) were then seeded on 6 well plate and culturedfor 1, 2 or 3 weeks in osteoblast differentiation medium (DMEM-HG, 10%FCS, 100 nM Dexamethasone, 0.05 mM Ascorbic acid, 10 mMβ-glycerolphosphate).

DETAILED DESCRIPTION OF THE INVENTION

According to the principles of the present invention, it is nowdisclosed that isolated FGF variant polypeptides of the presentinvention comprising amino acid truncations of the N-terminus adjacentto the β1 strand or extending into β1 strand of the core structure,while retaining the LYC motif, yield variants with improved properties,in addition to altered specificity to FGFRs. The variants thus obtainedwill have improved properties in terms of receptor specificity,stability and/or affinity in addition to enhanced mitogenic activityand/or differentiation potential. These variants may further compriseadditional modifications providing variants with improved stability,solubility or yield.

DEFINITIONS

For convenience certain terms employed in the specification, examplesand claims are described here.

The term “FGF2”, also known as basic FGF, bFGF, prostatin and heparinbinding growth factor 2, is highly conserved among species and has beenshown to stimulate the proliferation of a wide variety of cell types.The sequence of FGF2 has been disclosed in U.S. Pat. Nos. 4,994,559;5,155,214; 5,439,818 and 5,604,293. Human FGF2 is expressed in severalforms, a 210 aa precursor, a 157 aa form, a 155 aa form, a 146 aaN-terminal truncated form and several others (reviewed in Bikfalvi etal., 1997). The 155 aa form serves as the template for the truncatedpolypeptides disclosed herein. FGF2^(QQ) refers to an FGF2 ligand havingwild type activity towards all FGF receptor subtypes. As it isessentially indistinguishable from the wild type it has been used as areference in some experiments. FGF2^(QQ) has amino acid substitutions ofglutamine at positions 3 and 5 (Alanine 3 is replaced by glutamine,Ala3Gln; Serine 5 is replaced with glutamine, Ser5Gln).

Fibroblast growth factors (FGFs) constitute a large family ofstructurally related, heparin binding polypeptides, expressed in a widevariety of cells and tissues. Overall, the FGFs share between 17-72%amino acid sequence homology and a high level of structural similarity.A homology core of about 120 amino acids is highly conserved and hasbeen identified in all members of the family. Twelve antiparallelstrands have been identified in the core structure, labeled β1 throughβ12, linked one to another by loops of variable lengths, organized intoa trefoil internal symmetry. FIG. 1 in Mohammadi et al. (2005) showsamino acid sequence alignment of the β-trefoil strands for FGF1 throughFGF23.

The core domain of FGF2 extends from amino acid Lys30 to Lys154; thecore domain of FGF4 extends from amino acids Arg84 to Leu206.

The term “the N-terminal domain” refers to the amino acid residuesincluded in the N-terminus and adjacent to the β1 strand. The β1 strandof FGF2 consists of the amino acid sequence: KRLYCK (SEQ ID NO:92); the131 strand of FGF4 consists of the amino acid sequence: RRLYCN (SEQ IDNO:93). The Leu-Tyr-Cys (LYC) amino acid motif is a constant feature ofthe β1 strand of all wild type FGF ligands. Most of the wild type FGFligands have an LYC, LYS or LYT motif. As used herein and in the claimsthe term “beta1” or “β1” “β1 strand” refers to the most N-terminalantiparallel 13 strand of the core structure as disclosed herein. The β1strand is adjacent to the N-terminal amino acid sequence.

As used herein and in the claims the terms “amino terminus” and“N-terminus” of a polypeptide may be used interchangeably. Similarly,the terms “carboxy terminus” and “C-terminus” may be usedinterchangeably.

The beta8-beta9 loop of FGF2 consists of amino acids LESNNYNTY (SEQ IDNO:94; See FIG. 1A, underlined and labeled sequence).

The FGF ligands with increased receptor selectivity are useful in thetreatment of various pathological conditions including tissue repair andregeneration, wound and ulcer healing, bone and cartilage disorders,bone fracture healing, osteoporosis and other skeletal disorders. Otheruses include remodeling cardiac tissue and improving cardiac function,in particular for new blood vessel growth thereby providing analternative route for blood to bypass clogged and blocked arteries inthe heart.

Variant polypeptides are useful as site-specific carriers for deliveryand concentration of bioactive agents to cells, tissues, or organs inwhich a therapeutic effect is desired to be effected. These variants maybe therapeutically beneficial for treating skeletal disorders, includingbut not limited to achondroplasia, and proliferative diseases includingbut not limited to multiple myeloma, transitional cell carcinoma (TCC)of the bladder, breast cancer and cervical carcinoma. The targetingpolypeptides are fusion proteins, chimeric recombinants, hybrid proteinsor conjugates.

For pharmaceutical use the FGF variants of the present invention areformulated for administration via intra-articular, intravenous,intramuscular, subcutaneous, intradermal, or intrathecal routesaccording to conventional methods of use. The dosage will be prescribedaccording to common regimes in the art, while taking into considerationvariables such as: weight, age, location of injury, extent of injuryetc. The pharmaceuticals will include FGF variants of the presentinvention in addition to a carrier, namely, saline, buffered saline, 5%dextrose in water and alike. Additional excipients which prolong thehalf-life or biological activity of the active ingredients might also beadded, for instance: preservatives, solubilizers, buffering agents,hyaluronic acid, albumin etc. Other compounds which are known toincrease the resistance to proteolitic degradation might also be added.The treatment is suitable for an acute injury as well as for a chroniccondition requiring prolonged treatment.

For convenience certain terms employed in the specification, examplesand claims are described here.

As used herein and in the claims the term “FGFR” denotes a receptorspecific for FGF molecule(s), which is necessary for transducing thesignal, exerted by FGF to the cell interior, typically comprising anextracellular ligand-binding domain, a single transmembrane helix, and acytoplasmic domain that contains a tyrosine kinase activity. The term“FGFR” includes various isotypes of the receptors including solubleversions comprising the extracellular domain and lacking thetransmembrane and kinase domains.

As used herein and in the claims the term “weak FGF” denotes an FGFmolecule or variant which, after binding to an FGF receptor, elicitsstimulation of mitogenesis at most half that of the same cells exposedto the wild type parent FGF molecule, as measured in cell based assaysknown in the art. “Inactive FGF” denotes the variant that elicitsstimulation of mitogenesis at most one tens that of the same cellsexposed to the wild type parent FGF molecule.

As used herein and in the claims the term “an isolated FGF polypeptidehaving increased receptor selectivity” denotes an isolated FGFpolypeptide molecule, having either enhanced or reduced biologicalactivity toward at least one but not all FGFR, compared to thecorresponding wild type FGFR. The biological activity toward at leastone receptor, but not all FGF receptors, is reduced or increased by afactor of at least two. In some embodiments the biological activitytoward at least one receptor, but not all FGF receptors, is reduced orincreased by a factor of at least four, at least five, at least seven orat least ten.

Biological activity can be measured by methods known in the art. In someembodiments biological activity is measured as cell proliferation and/orsubstrate phosphorylation.

The term “affinity” as used herein denotes the ability of a ligand orvariant of said ligand to bind to a specific receptor. Modifications toa ligand that stabilize favorable conformation or enhance amino acidside chain interactions will result in increased receptor affinity whilethose, which destabilize favorable conformation or decrease amino acidside chain interactions will result in decreased receptor affinity. Acompetitive binding assay was established to determine the relativeaffinity of isolated FGF polypeptides compared to that of wild typeparent FGF towards an FGF receptor.

As used herein the term “differentiation factor” refers to a substance,in particular a polypeptide, which determines the fate that a cell willacquire upon exposure to that substance, alone or in combination withother substances. In a non-limiting example, differentiation isdetermined by morphological and phenotypic changes or by biochemical ormolecular changes.

As used herein the term “mitogen” or “proliferation factor” refers to asubstance that induces an increase in the number of cells.

As used herein and in the claims the term “core”, “core domain” or “corestructure” denotes a region of homology of about 120 amino acids that isfound in all native FGFs. Twenty eight amino acid residues are highlyconserved and four are identical. Twelve structurally conservedanti-parallel β strands have been identified within the core domain inall the FGFs. The core domain further comprises the FGFR- andheparin-binding sites. Sequence alignment and location and length of theβ strands for FGF-1 through FGF-23 is depicted in FIG. 1 of Mohammadi etal. (2005). The amino acid sequences of the core structure of FGF2 andFGF4 are depicted herein in FIG. 1A.

“Nucleic acid sequence” or “polynucleotide” as used herein refers to anoligonucleotide or nucleotide and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin, which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,“amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring, synthetic or recombinant molecules. The termslisted herein are not meant to limit the amino acid sequence to thecomplete, wild type amino acid sequence associated with the recitedprotein molecule.

The term “variant” as used herein refers to a polypeptide sequence thatpossesses some modified structural property of the wild type or parentprotein. For example, the variant may be truncated at either the aminoor carboxy terminus or both termini or may have one or more amino acidsdeleted, inserted and or substituted. The most preferred method forproducing the variants is through recombinant DNA technologies, wellknown to those skilled in the art. For example, the variants may beprepared by Polymerase Chain Reaction (PCR) using specific primers foreach of the truncated forms or the amino acid substitutions as disclosedherein below. The PCR fragments may be purified on an agarose gel andthe purified DNA fragment may be cloned into an expression vector andtransfected into host cells. The host cells may be cultured and theprotein harvested according to methods known in the art.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of amplifying nucleic acids, as disclosed in U.S. Pat. Nos.4,683,195; 4,683,202 and 4,965,188 hereby incorporated by reference.

The term “expression vector” and “recombinant expression vector” as usedherein refers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. The expression vector may comprise sequences encodingheterologous domains including but not limited to protein detection,purification or cleavage sequences that may be fused at the N- orC-terminus to the desired coding sequence, to yield a fusion protein.The present invention encompasses expression vectors that are integratedinto host cell genomes, as well as episomal vectors.

As used herein, the “amino acids” used in the invention are those thatare natural, those that are available commercially or are available byroutine synthetic methods. Certain amino acid residues may requirespecial methods for incorporation into the peptide, and sequential,divergent or convergent synthetic approaches to the peptide sequence areuseful in this invention. Natural coded amino acids and theirderivatives are represented by either the one-letter code orthree-letter codes according to IUPAC conventions. When there is noindication, the L isomer is used.

As used herein and in the claims a “bioactive agent” is any agent whichis desired to be delivered to cells, tissues or organs for modulating ormodifying cell function, including for therapeutic effects. Inaccordance with the present invention, bioactive agents include, but arenot limited to, pharmaceutically active compounds or diagnosticcompounds. These include, but are not limited to, peptides and peptideanalogs, peptidomimetics, oligopeptides, proteins, apoproteins,glycoproteins, antigens and antibodies or antibody fragments thereto,receptors and other membrane proteins, aptamers, enzymes, coenzymes,enzyme inhibitors, amino acids and their derivatives, hormones, lipids,phospholipids, liposomes; toxins; tyrosine kinase inhibitors,photoreactive agents, antibiotics; analgesics and anti-inflammatorysubstances; antimicrobial agents; antihypertensive agents; antiviralagents; antihistamines; anti-cancer drugs including chemotherapeuticagents; tranquilizers; neuroprotective agents; antispasmodics;anti-Parkinson agents; vitamins. Other bioactive agents includenucleotides; oligonucleotides; polynucleotides; and their biologicallyfunctional analogs and derivatives; plasmids, cosmids, artificialchromosomes, other nucleic acid vectors; antisense polynucleotidesincluding those substantially complementary to at least one endogenousnucleic acid; promoters; enhancers; inhibitors; other ligands forregulating gene transcription and translation.

“Bone defect” or “bone disorder” may result from mutation, fracture,from surgical intervention, from a proliferative disease such asmultiple myeloma or metastases, of from dental or periodontal disease.By “cartilage defect” or “cartilage disorder” is meant cartilage thathas been damaged by disease, injury or trauma. Contemplated areindications including rheumatoid arthritis, osteoarthritis and jointinjuries.

Representative uses of the compounds of the present invention include:repair of bone defects and deficiencies, such as those occurring inclosed, open and non-union fractures; prophylactic use in closed andopen fracture reduction; promotion of bone and cartilage healing inplastic surgery; stimulation of bone ingrowth into non-cementedprosthetic joints and dental implants; elevation of peak bone mass inpre-menopausal women; treatment of growth deficiencies; treatment ofperiodontal disease and defects, and other tooth repair processes;increase in bone formation during distraction osteogenesis; treatment ofarticular chondrocytes prior to transplantation and treatment of otherskeletal disorders, such as age-related osteoporosis, post-menopausalosteoporosis, glucocorticoid-induced osteoporosis or disuse osteoporosisand arthritis. Certain variants may be useful in other tissueregeneration and repair indications, for example in liver, pancreas,nerve regeneration and repair. The compounds of the present inventionare useful in repair of congenital, trauma-induced or surgical resectionof bone (for instance, for cancer treatment), and in cosmetic surgery.Treatment includes direct application of the variants to the traumatizedarea or systemic therapy as well as treatment of cells ex vivo and invitro for tissue engineering and tissue regeneration. Additionally thevariants may be provided in cell therapy.

As used herein, the terms “fusion protein” or “chimera”, “chimericrecombinant” or “hybrid” refer to a single polypeptide produced usinghost cells expressing a single polynucleotide encoding an isolated FGFpolypeptide of the invention and a bioactive agent including apolypeptide, peptide or peptide analog contiguous and in open readingframe. Certain peptide linkers may separate the FGF and the bioactivepolypeptide, peptide or peptide analog. The present invention provides ahighly effective system for delivery of an activity-inducing moiety intoa particular type or class of cells. The fusion proteins generated canbe screened for the desired specificity and activity utilizing themethods set forth in the examples and by various routine procedures. Asused herein, the term “conjugate” refers to a bioactive agent covalentlybound to a carrier or targeting moiety. Certain variants of theinvention provide carriers or targeting agents for bioactive agents. AnFGF “targeting molecule” or “complex molecule” refers to an isolated FGFpolypeptide of the invention linked to a bioactive agent.

Provided in the present invention are pharmaceutical compositionscomprising an isolated FGF polypeptide and a bioactive agent as a fusionprotein or alternatively an isolated FGF polypeptide conjugatecomprising an isolated FGF polypeptide and a bioactive agent useful forFGF targeting. The present invention further provides methods forinhibiting proliferation of cells that express FGFRs comprisingadministering an FGF variant targeting composition to the cells. Forexample, the composition is administered in a therapeutically effectiveamount to a subject that has a tumor, wherein the tumor cells expressspecific FGFR.

FGF activity is conveniently determined using biological assaysperformed in-vitro, ex-vivo and in vivo. The assays are used todemonstrate the activity elicited upon binding of an FGF molecule to itsreceptors. The biological assays routinely used to test activities ofvariant FGFs include, but are not limited to, the following:

binding of variant FGFs to cloned FGF receptors expressed onimmortalized cell lines, thereby eliciting a biological responseincluding cell proliferation or inhibition of cell proliferation;

cell proliferation and differentiation in cell culture systems;

phosphorylation assays;

stimulation of bone growth in animal models of bone growth and cellcultures; enhancement of cartilage repair in animal models of cartilagedisease and trauma.

Polynucleotide Sequences

The present invention also provides for an isolated nucleic acidmolecule, which comprises a polynucleotide sequence encoding the proteinof the invention and a host cell comprising this nucleic acid molecule.Furthermore, also within the scope of the present invention is a nucleicacid molecule containing a polynucleotide sequence having at least 90%sequence identity, preferably about 95%, and more preferably about 97%identity to the above encoding nucleotide sequence as would wellunderstood by those of skill in the art.

The invention also provides isolated nucleic acid molecules thathybridize under high stringency conditions to polynucleotides having SEQID NO:34-59 or SEQ ID NO:69-77 or SEQ ID NO:91 or the complementthereof. As used herein, highly stringent conditions are those which aretolerant of up to about 5-20% sequence divergence, preferably about5-10%. Without limitation, examples of highly stringent (−10° C. belowthe calculated Tm of the hybrid) conditions use a wash solution of0.1×SSC (standard saline citrate) and 0.5% SDS at the appropriate Tibelow the calculated Tm of the hybrid. The ultimate stringency of theconditions is primarily due to the wash conditions, particularly if thehybridization conditions used are those which allow less stable hybridsto form along with stable hybrids. The wash conditions at higherstringency remove the less stable hybrids. A common hybridizationcondition that can be used with the highly stringent to moderatelystringent wash conditions described above may be performed byhybridizing in a solution of 6×SSC (or 6×SSPE), 5×Denhardt's reagent,0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA at anappropriate incubation temperature Ti. See generally Sambrook et al.(1989) for suitable high stringency conditions.

Stringency conditions are a function of the temperature used in thehybridization experiment and washes, the molarity of the monovalentcations in the hybridization solution and in the wash solution(s) andthe percentage of formamide in the hybridization solution. In general,sensitivity by hybridization with a probe is affected by the amount andspecific activity of the probe, the amount of the target nucleic acid,the detectability of the label, the rate of hybridization andhybridization duration. The hybridization rate is maximized at a Ti(incubation temperature) of 20-25° C. below Tm for DNA:DNA hybrids and10-15° C. below Tm for DNA:RNA hybrids. It is also maximized by an ionicstrength of about 1.5M Nat⁺. The rate is directly proportional to duplexlength and inversely proportional to the degree of mismatching.Specificity in hybridization, however, is a function of the differencein stability between the desired hybrid and “background” hybrids. Hybridstability is a function of duplex length, base composition, ionicstrength, mismatching, and destabilizing agents (if any).

The Tm of a perfect hybrid may be estimated for DNA:DNA hybrids usingthe equation of Meinkoth, as

Tm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L

and for DNA:RNA hybrids, as

Tm=79.8° C.+18.5(log M)+0.58(% GC)−11.8(% GC)²−0.56(% form)−820/L

where M, molarity of monovalent cations, 0.01-0.4 M NaCl,

-   -   % GC, percentage of G and C nucleotides in DNA, 30%-75%,    -   % form, percentage formamide in hybridization solution, and    -   L, length hybrid in base pairs.

Tm is reduced by 0.5-1.5° C. (an average of 1° C. can be used for easeof calculation) for each 1% mismatching. The Tm may also be determinedexperimentally.

Filter hybridization is typically carried out at 68° C., and at highionic strength (e.g., 5-6×SSC), which is non-stringent, and followed byone or more washes of increasing stringency, the last one being of theultimately desired high stringency. The equations for Tm can be used toestimate the appropriate Ti for the final wash, or the Tm of the perfectduplex can be determined experimentally and Ti then adjustedaccordingly.

The present invention also relates to a vector comprising the nucleicacid molecule of the present invention. The vector of the presentinvention may be, e.g., a plasmid, cosmid, virus, bacteriophage oranother vector used e.g. conventionally in genetic engineering, and maycomprise further genes such as marker genes which allow for theselection of said vector in a suitable host cell and under suitableconditions.

The use of FGFs and FGF fragments for targeting cytotoxic agents hasbeen disclosed in WO 01/39788 and U.S. Pat. Nos. 5,191,067; 5,576,288;5,679,637. A mitogenically active FGF molecule provides a route forintroducing the selected agents into the cell.

Pharmacology

The present invention also provides for a composition comprising atleast one polypeptide of the present invention. “Therapeutic” refers toany pharmaceutical, drug or prophylactic agent which may be used in thetreatment (including the prevention, diagnosis, alleviation, or cure) ofa malady, affliction, disease or injury in a patient. Therapeuticallyuseful peptides, polypeptides and polynucleotides may be included withinthe meaning of the term pharmaceutical or drug.

The term “excipient” as used herein refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofa compound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.Pharmaceutical compositions may also include one or more additionalactive ingredients.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

The pharmaceutical composition of this invention may be administered byany suitable means, such as orally, intranasally, subcutaneously,intramuscularly, intravenously, intra-arterially, or parenterally.Ordinarily, intravenous (i.v.) or parenteral administration will bepreferred.

METHODS OF THE PRESENT INVENTION

According to another aspect of the present invention it is disclosedthat the preferred variant FGFs have improved therapeutic utility indiseases and disorders involving FGF receptors. For example, the FGF2 orFGF4 variant promotes fibroblast growth factor activity in a cell andenhances a biological process associated with fibroblast growth factoractivity. Biological activity may be selected from the group consistingof: promotion of cell proliferation, induction or repression of terminaldifferentiation in a cell, promotion of angiogenesis, promotion of woundhealing, promotion of chondrogenesis or osteogenesis, and promotion ofneurogenesis. Certain FGF variants of the present invention may be usedin the treatment of myocardial infarction (For example U.S. Pat. No.4,296,100 and U.S. Pat. No. 4,378,347), treating degenerativeneurological disorders, including Alzheimer's disease and Parkinson'sdisease, promoting angiogenesis, promoting bone healing, and promotingmuscle healing. Additional therapeutic uses include surgical ornon-surgical administration of angiogenic FGF polynucleotides andpolypeptides for treating coronary and peripheral vascular disease.

An FGF ligand having increased receptor selectivity is expected toreduce the severity of side effects associated with ligands recognizingand activating multiple FGF receptor subtypes.

Accordingly the present invention provides a method of treating anindividual in need thereof comprising the step of administering to thatindividual a pharmaceutical composition comprising a therapeuticallyeffective amount of at least one N-terminal truncated FGF2 and or FGF4polypeptide variant according to the present invention.

In another aspect the present invention provides a method of inducingcellular expansion, comprising the steps of:

-   -   a. isolating a population of cells to be expanded; and    -   b. exposing said cells to at least one N-terminal truncated FGF2        or FGF4 polypeptide variant according top the principles of the        present invention.

The principles of the invention are demonstrated by means of thefollowing non-limitative examples.

EXAMPLES Example 1 Preparation of FGF Polynucleotide and PolypeptideVariants

The FGF variants were prepared by standard PCR amplification and cloninginto a p89BS expression vector. The details of the vector are disclosedin PCT publication WO 02/022779, of some of the inventors of the presentinvention. The vector has unique NdeI-BamHI restriction sites and thePCR-generated DNA fragments encompassing the coding region of a numberof proteins and variants were produced having NdeI and BamHI sites atthe 5′ and 3′ ends respectively. The polynucleotide fragments encodingthe proteins were ligated into digested p89BS and the plasmid was usedto transform E. coli cells, such as JM109, TG1, TG2, DHα, and XL1blue,using standard transformation protocols.

The primers used for construction of the variants are shown in Table IV,herein below.

TABLE IV forward and reverse primers used in preparation of the FGF2 and FGF4 polynucleotide variants of the present  invention. Poly-nule- otide SEQ ID NO: Forward Primer Backward Primer FGF2- 34 5′GGAATTCCATATGCACTTCAAG 5′CGGGATCCTCAGCTCTTAG Δ24 GACCCCAAG CAGSEQ ID NO: 78 SEQ ID NO: 88 FGF2- 35 5′GGAATTCCATATGTTCAAGGACSEQ ID NO: 88 Δ25 CCCAAGCG SEQ ID NO: 79 FGF2- 36 5′GGAATTCCATATGAAGGACCCC SEQ ID NO: 88 Δ26 AAGCGGCTG SEQ ID NO: 80 FGF2-41 5′ GGAATTCCATATGCTG SEQ ID NO: 88 Δ31 TACTGCAAAAACGGGGGCSEQ ID NO: 81 FGF4- 69 5′GGAATTCCATATGGCCGGCGAC 5′CGGGATCCTCACAGCCTGGΔ72 TACCTGCTGG GGAGGAAGTG SEQ ID NO: 82 SEQ ID NO: 89 FGF4- 70 5′GGAATTCCATATGGGCGACTAC SEQ ID NO: 89 Δ73 CTGCTGGGCATC SEQ ID NO: 83FGF4- 71 5′GGAATTCCATATGGACTACCTGC SEQ ID NO: 89 Δ74 TGGGCATCAAGSEQ ID NO: 84 FGF4- 72 5′GGAATTCCATATGTACCTGCTGG SEQ ID NO: 89 Δ75GCATCAAGCG SEQ ID NO: 85 FGF4- 73 5′GGAATTCCATATGCTGCTGGGC SEQ ID NO: 89Δ76 ATCAAGCGGC SEQ ID NO: 86 FGF4- 74 5′GGAATTCCATATGCTGGGCATCSEQ ID NO: 89 Δ77 AAGCGGCTGC SEQ ID NO: 87 FGF4- 75 Basilico, et al. Δ78

A single colony was selected and grown overnight (ON) at 37° C. in atwo-liter flask containing 330 ml of TB125 medium (Tryptone15 gr/L,Yeast extract 30 gr/L, KH₂PO₄ 2.31 gr/L, K₂HPO₄ 12.5 gr/L, Glycerol 5gr/L) supplemented with 200 μg/ml ampicillin. The bacterial suspensionwas centrifuged at 4000 rpm (4° C.) for 15 minutes, and the medium wasdiscarded. The bacterial pellet was then suspended in 25 ml of 1×PBSbuffer containing protease inhibitors, sonicated on ice, and centrifugedat 10,000 rpm (4° C.) for 15 minutes. The protein supernatant wascollected and 3 ml of heparin-Sepharose® beads slurry was added andshaken gently for 6 hours at 4° C. The beads were loaded onto a column,washed extensively with PBS buffer containing 0.3M NaCl, and eluted in 7ml PBS containing 2-2.5M NaCl. The FGF variant proteins were thendialyzed against 1×PBS and repurified on FPLC using a heparin Sepharosecolumn (HiTrap™Heparin, Amersham Pharmacia biotech) with a 0-2.5M NaCl(in PBS-0.05% CHAPS) linear gradient in the same dialysis buffer. Thepurified proteins were later stored at −70° C.

Example 2 FGF Variant Binding to FGFR-Transfected FDCP Cell Lines

The FDCP cell line is a murine immortalized, interleukin 3(IL-3)-dependent cell line of myelocytic bone marrow origin that doesnot express endogenous FGF Receptors (FGFR). Upon transfection with FGFRcDNA, the FDCP cell line exhibits a dose-dependent proliferativeresponse to FGF that can replace the dependence on IL-3. FGFRtransfected FDCP cells can therefore be used to screen variant FGFs forspecific inhibitors, activators or for FGFR signaling. FDCP cellsresponse to various ligands is quantitated by a cell proliferation assaywith XTT reagent (Cell Proliferation Kit, Biological Industries Co.).The method is based on the capability of mitochondrial enzymes to reducetetrazolium salts into a colorogenic compound, which can be quantitatedand is indicative of cell viability.

Specifically, FDCP cells stably expressing FGFR4, FGFR3—IIIc, FGFR3—IIIbisoforms, FGFR2IIIc or FGFR1IIIc were grown in “full medium” (Iscove'sMedium containing 2 ml glutamine, 10% FCS, 100 μg/ml penicillin, 100μg/ml streptomycin) supplemented with 5 μg/ml heparin and 10 ng/ml FGF.Cells were split every 3 days and kept in culture for up to one month.One day prior to the experiment the cells were split. Before theexperiment the cells were washed 3 times (1000 rpm, 6 min) with fullmedium. The cells were resuspended and counted with Trypan Blue. Twentythousand (2×10⁴) cells were added to each well of 96-well plate in 50 μlfull medium containing heparin. Conditioned medium containing FGF wildtype parent or variants at varying concentrations with heparin was addedin an additional volume of 50 μl full medium to bring the final volumeto 100 μl. The plate was incubated for 48 hours at 37° C. To assay cellproliferation, 100 μl of PMS reagent was added to 5 ml of XTT reagentand mixed well (according to manufacturer's protocol). Fiftymicro-liters (50 μl) of the latter solution were added into each well,and the plates incubated at 37° C. for 4 hours and the color was read bya spectro-ELISA reader at A_(490nm).

In these experiments FDCP cells expressing FGFR4, the FGFR3 isoformsFGFR3IIIb and FGFR3IIIc, FGFR2 or FGFR1 were grown in the presence ofvarying concentrations of the polypeptides of the invention

Example 3 Effect of Variants on Growth Arrest of RCS Chondrocytes

RCS is a rat chondrosarcoma derived cell line expressing preferentiallyhigh levels of FGFR2 and FGFR3 and low levels of FGFR1. In this cellline, FGFR functions as an inhibitor of cell proliferation similar toits expected role in the achondroplasia phenotype. In order to inhibitcell proliferation, the variants have to specifically induce FGFR signaltransduction allowing the measuring of FGF affinity and specificity tothe FGFRs reflected by the concentration dependence of induced growtharrest.

The screening was performed on RCS cells in 96 well plates. Cells wereseeded at a concentration of about 2,000 cells/well. The following day10 ng/ml FGF or FGF variants and 5 μg/ml heparin were added to thecells. Positive and negative controls for cell proliferation areincluded in this assay at the same concentrations as the testedcompounds. On the fourth day of incubation, plates were observed underthe microscope. If all cells were viable, no quantitative assay tomeasure the effect of the variants was performed. If cell death wasobserved, the Cy-Quant® assay kit is used to measure the amount of thecells. The results are measured in a fluoro ELISA reader.

Example 4 JNK Activation by FGF Variants

JNK activation by wild type and variant FGF2 proteins is determined inan in vitro cell assay using stably transfected RCJ (Rat calvaria) celllines expressing in an inducible manner either the FGFR1IIIC, 2IIIC,3IIIC or 4 isoforms. In summary, RCJ over-expressing FGFR subtypes aregrown in α-MEM⁺⁺ medium (15% FCS, G-418 600 μg/ml, hygromycin andtetracycline 2-3 μg/ml). The concentration of cells seeded rangesbetween 7.5×10⁴ and 6×10⁵ cells/well in a 6 well plate. The cell mediumis removed 14-16 hours prior to beginning of experiment. Four to fivehours before addition of the FGF ligand or variants, the cells areserum-starved. Either wild type or variant FGF are added at aconcentration range of 0.1-100 ng/ml for 5-7 minutes at 37° C. FGFstimulation is stopped by cooling the cells on ice followed by washing 3times with cold PBS. The cells are lysed by addition of lysis buffer (1mM EGTA, 1 mM EDTA, 25 mM Tris/50 mM Hepes, 25 mM NaF, 50 mMβ-glycerophosphate, 50 mM NaCl, 10% Glycerol, 1% NP40, pH 7.5, freshlyprepared Orthovanadate to 2 mM, and protease inhibitors) for 10 minuteson ice. The cell lysates are collected and spun for 10 minutes at 15,000rpm. Protein aliquots are loaded on SDS-PAGE and activation is viewed ina standard Western assay using rabbit anti-active JNK antibodies(Promega). In each lane, cell lysate of RCJ cells exposed to 0, 12.5,25, 50 or 100 ng of wild-type or variant FGF is loaded.

Example 5 Phosphorylation Assay Using Anti-MAPK/ERK Antibodies

FGF/FGFR-dependent ERK activation signal transduction is measured in anELISA assay using monoclonal anti-diphosphorylated ERK antibodies. Theassay is followed by reading A_(450nm) after addition of the TMB reagentto monitor the total ERK activation.

RCJ over-expressing FGFR subtypes were grown as described hereinabove inExample 4. Following the collection of cell lysates, SDS was added tothe supernatants to 1.5% final concentration and the mixture incubatedfor 15 min at room temperature. Following protein determination, theprotein and SDS concentrations were adjusted with lysis buffer to afinal concentration of 7 μg protein and 0.15% SDS in 100 μl. 100 μl ofsample lysate is added to a Maxisorp® 96 well plate (Nuncimmuno-plate430341) precoated with monoclonal anti-diphosphorylatedMAPK/ERK (Sigma M8159) diluted to 1:3000 with a mixture 4% BSA in TBSTand lysis buffer adjusted to 0.15% SDS. The plates are incubated,shaken, for 2 hours at room temperature. The wells were washed and eachwell incubated with 100 μl of 1:10,000 dilution of HRP-conjugated goatanti-mouse IgG (Jackson Immunoresearch 800-367-5296) in 2% BSA/TBST for1-1.5 hrs at room temp, with shaking. Following incubation, the sampleswere washed 5-6 times with TBST, and 1000 of developing medium (1:1mixtures A and B of ImmunoPure® TMB substrate kit) is added for 10minutes at room temperature. The reaction was stopped by the addition ofH₂SO₄ and the absorbance was read at 450 nm.

Example 6 Chondrocyte Expansion

The effect of FGF and FGF variants on proliferation of articularchondrocytes was tested. Articular Chondrocyte culture: Chondrocytes areisolated from pig or human biopsies and cultured using the FGF variantsof the present invention to identify the effect of the variants onproliferation and differentiation. The procedure employed for theisolation and propagation of articular chondrocytes is presented below.

Reagents:

-   -   Dulbecco's MEM (DMEM) (Gibco BRL)    -   MEM Non-Essential Amino Acids (Gibco BRL)    -   Sodium Pyruvate (Gibco BRL)    -   Fetal Bovine Serum (FBS) (Gibco BRL)    -   Streptomycin, Penicillin, Nystatin Solution (Biological Indus.        Ltd.)    -   Trypsin-EDTA (Gibco BRL, cat. no. T8154) or Versene-Trypsin (Bio        LAB Ltd.)    -   Collagenase Type 2 (Worthington Biochem. Corp.). A stock        solution of 1700 units/ml Collagenase in DMEM was prepared and        filtered (0.2 μm).    -   EZ-RNA kit (Biological Industries Israel Beit Haemek)    -   Reverse-iT strand synthesis kit (AB gene)    -   GeneAmp® 5700 Sequence detection system (Applied Biosystems)    -   RPLPO Assays-on demond™ (Applied Biosystems)    -   Preparation of FBS-DMEM medium:

FBS (50 ml), 5 ml of antibiotic solution, 5 ml Sodium Pyruvate, 5 ml MEMnon-essential amino acids were added to a 500 ml bottle of DMEM. Wherespecified, FGF2, FGF4 or FGF variants were added to a finalconcentration of 10 ng/ml.

Isolation of Cells from Cartilage Biopsy:

A piece of cartilage tissue is minced into 2 to 4 mm pieces with asterile scalpel. The collagenase solution is diluted 1:4 in FBS-DMEM,added to the tissue sample and left to incubate on a rotator at 37° C.,overnight (ON). The cells are centrifuged (1200 rpm, 5-10 min). Themedium is aspirated, the cells washed in 5 ml medium and recentrifuged.The cells are resuspended in culture medium and seeded in 25 cm² or 75cm² flasks at a concentration of approximately 1×10⁶ cells per flask.The cells were incubated in a 5% CO₂ incubator at 37° C. The cell mediumwas replaced every 2-3 days.

Procedure for passaging cells (trypsinization):

When the cell culture reached the desired confluency the medium wasremoved and the cells trypsinized in the following manner: Onemilliliter (1 ml) of the trypsin solution was added to a 25 cm² flask or2.5 ml to a 75 cm² flask. The flask was gently shaken by hand for twoseconds and the trypsin solution was immediately removed. Another 1 mlof trypsin was added to a 25 cm² flask or 2.5 ml to a 75 cm² flask. Theflask was gently shaken by hand for ˜30 seconds and left to incubate at37° C. for 3-5 minutes. Verify under the microscope that cells aredetached. The trypsin was neutralized by adding FBS-MEM; add 10 ml to a25 cm² flask and 25 ml to a 75 cm² flask. The cells were split to 2-3new flasks and 20 ml fresh pre-warmed medium was added. The expansion ofcells and trypsinization was performed as necessary.

Furthermore, the cell population grown on the above matrices expressesseveral of the chondrocyte differentiation markers. One of severalphenotypes expressed during chondrocyte differentiation isglycosaminoglycan (GAG) production. The production of GAGs is identifiedin histological staining using Alcian blue and quantitated using the DMB(3,3′-dimethoxybenzidine dihydrochloride) dye method.

Proliferation of the cartilage cells in the presence of the differentvariants can be quantitated by one of two methods, CyQUANT® (MolecularProbes) or XTT reagent (Biological Industries, Co.). Human or porcinearticular chondrocytes (10⁴-10⁶ cells/30-1000 are grown in the presenceof the variants of the invention in microwell plates. The cells aregrown overnight in MEM, collected and XTT reagent is added for 3-4 hoursand the plates read in an ELISA reader at A₄₉₀ nm.

Example 7 Effect of FGF Variants on Bone Fracture Healing

Ulnas are fractured in New Zealand Rabbits in compliance with the AnimalCare Committees. The ulna was chosen because it is only slightlyweight-bearing and allows the creation of a bone defect withoutrequiring a cast or other immobilization treatment. In addition, thisgap constitutes a spontaneously healing defect that allows theevaluation of the tested agent. The primary indices of fracture healingare accelerated duration of healing and callus formation.

A 0.6 cm radial gap osteotomy is created under anesthesia with rotarysaw in both ulnas of each animal. Approximately 1 ml of a compositioncomprising the test compounds is administrated by injection into thegap. The periosteum, which is not resected during the surgery, is usedto close the gap. Fracture healing is radiologically evaluated everyweek up to 4 weeks p.o. (post osteotomy). An X-ray closure of both limbsin a lateral position was taken. X-ray films are examined by a FilmDigitizer, and the following parameters were measured: Total area ofregenerated bone appearing around and within the bone gap defect (callusarea) and the relative density of the newly regenerated bone in the gapdefect. Histopathlogical evaluation was made by preparing thin sectionsthat were stained with hematoxylin and eosin for cytoplasm and nucleus.Indigo-Carmin staining is also applied for detection of new generatedcallus.

Example 8 Bone Marrow Stem Cells: Isolation and Proliferation

Human articular chondrocytes were isolated from pieces of cartilageusing digestion enzymes. Isolated primary chondrocytes were seeded in 75cm² flasks (5×10⁴ cells) containing DMEM/F12, 20% human serum, 10 ng/mlof different FGF variant of the present invention and 5 μg/ml lowmolecular heparin (LMW). Cells were counted in different time pointsusing the Vi-Cell XR.

Example 9 Bone Marrow Stem Cells: Isolation and Expansion

Samples of human bone marrow were used to isolate mesenchymal stem cellsby adherence to plastic dishes in the presence of MSC expansion medium,namely, DMEM-LG containing 20% human serum (HS), or 10 ng/ml FGF2 andFGF4 variants of the present invention and with or without 5 mg/ml lowmolecular weight (LMW) Heparin. For proliferation assay 2×10⁴ cells wereseeded per well in a 24 wells plate. Cells were counted every 6-7 daysusing the Vi-Cell XR and reseeded to obtain several time points.

Example 10 Bone Marrow Stem Cells: In-Vitro Chondrogenesis Assay

The expanded chondrocytes or hBM-MSC were used for in-vitrochondrogenesis assay to produce high density pellet cultures. Therepellet cultures were incubated for 21 days in chondrocytesdifferentiation medium containing DMEM, 10 ng/ml TGF-b, 100 mMDexamethasone, 0.28 mM Ascorbic acid, 1 mM Sodium pyruvate, 40 μg/mlProline, 10 μg/ml Bovine insulin, 5.5 μg/ml Human transferin, 5 μg/mlSodium selenite, 0.5 mg/ml Bovine serum albumin and 1.7 μg/ml Linoleicacid.

Example 11 Bone Marrow Stem Cells: Histology Analysis of Pellet Cultures

Pellet cultures incubated for 3 weeks in differentiation medium werefixed in PFA solution and paraffin block were prepared. Sections wereused for analysis of proteoglycans by Alcian blue (AB) and Safranin O(SO) stains and for examine Col II expression by immunohistochemistrywith anti Col II antibodies.

Example 12 Bone Marrow Stem Cells: qPCR Analysis of Col II

RNA was isolated from pellet cultures by EZ-RNA kit (BiologicalIndustries Israel Beit Haemek). The RNA was used to prepare cDNA usingthe Reverse-iT strand synthesis kit (AB gene). qPCR was performed usingGeneAmp® 5700 Sequence detection system (Applied Biosystems). cDNAsamples were analyzed for quantitative expression of Col II and thereference gene RPLPO using Assays-on demond™ (Applied Biosystems). Foreach cDNA sample, the threshold cycle (Ct) value of target sequence wassubtracted by Ct value of RPLPO, to derive ΔCt. Relative level of Col IIwas calculated as 2ΔCt.

Example 13 Bone Marrow Stem Cells: In-Vitro Osteogenesis Assay

Cells (2×10⁴ per well) were seeded on 6 well plate and cultures for 1, 2or 3 weeks in osteoblast differentiation medium (DMEM-HG, 10% FCS, 100nM Dexamethasone, 0.05 mM Ascorbic acid, 10 mM β-glycerolphosphate).Cells were stained with Alizarin Red for analysis of calcium in matrix.

Example 14 Effects of FGF Variants on Femoral Growth

Femoral bone cultures are performed by excising the hind limbs of wildtype mice. The limbs are carefully cleaned from the surrounding tissue(skin and muscles) and the femora exposed. The femora are removed andfurther cleared from tissue remains and ligaments. The femora aremeasured for their initial length, using a binocular with an eyepiecemicrometer ruler. The bones are grown in 1 ml of medium with FGF2 orFGF4 variants in a 24 well tissue culture dish. The growing medium isa-MEM supplemented with penicillin (100 units/ml), streptomycin (0.1mg/ml) and Nystatin (12.5 units/ml). In addition, the medium containsBSA (0.2%), β-glycerophosphate (1 mM) and freshly prepared ascorbic acid(50 μg/ml). The bones are cultured for 15 days. Measurements of bonelength and medium replacement are performed every three days. At the endof the experiment, the growth rate of the bones are determined from theslope of a linear regression fit on the length measurements obtainedfrom day 3 to 12. Units given can be converted to length, 40 units=1 mm.

Example 15 Effect of FGF2 Variants in Bone Fracture Healing

Suitable animal models are used to create bilateral osteotomies todemonstrate the efficacy of the novel variants of the present invention.In a rabbit model a 6 mm osteotomy is created in New Zealand Rabbits incompliance with the Animal Care Committee of the Hebrew University. Theulna was chosen because it is only slightly weight-bearing and allowsthe creation of a bone defect without requiring a cast or otherimmobilization treatment. In addition, this gap constitutes aspontaneously healing defect that allows the evaluation of the testedagent. The primary indices of fracture healing are accelerated durationof healing and callus formation. The test compounds consist of FGF2 orFGF4 variants in a polymeric scaffold which facilitates bone growth.

Surgical Procedure:

Animals are anesthetized according to standard protocol. Gap formationis performed in the mid Ulna bone. A standard volume of 0.2 ml oftreatment formulation is put into the gap area in each limb and thefracture is closed. Animals are treated with analgesics for 3 days postoperation. The duration of the experiment is 6 weeks.

Healing Time and Quality Assessment:

Healing time evaluation: X-ray grading provides fracture healing statusassessment. Rabbits are x-rayed every other week for 5 weeks aftersurgery. Two orthopedic surgeons score X-rays in a blinded manneraccording to standard grading scale protocol.

Quality Evaluation:

At the end of the experiment rabbits are sacrificed and fracture area issent for histological and mechanical strength evaluation. Histology isscored by a pathologist for evaluation of histological changes duringthe healing process using standard staining methods, using hematoxylinand eosin for cytoplasm and nucleus. Indigo-Carmin staining is alsoapplied for detection of newly generated callus. Mechanical strengthevaluation is performed using the “4 points bending” method.

The Treatments Groups are:

Osteotomy without treatment, Osteotomy treated with polymeric scaffoldalone, Osteotomy treated with scaffold containing FGF2 and an osteotomytreated with scaffold containing FGF2 variants.

Example 16 Efficacy of FGF Variants in Distraction Osteogenesis

Distraction osteogenesis is a useful method for bone elongation ofextremities in short stature and for the treatment of extensive bonedefects. Several procedures for bone lengthening have been developed foruse in the clinic. The problems encountered in using this techniqueinclude an extended healing time and complications such as non-union orpoor quality of the regenerated bone.

The maximal rate of elongation used in the current procedure of limbelongation, while maintaining proper bone healing and reconstitution, is1 mm/day. Faster elongation rates have resulted in lack of fusion or inthe formation of weak bone that breaks easily or cannot bear bodyweight. In this process, extreme conditions of elongation (1.5 mm/day)will be performed in order to observe a more significant effect of theadded compounds on the background of natural healing.

The objectives of the experiment are to assess the quality of boneformation, time of bone formation and safety after elongation using acalcium phosphate (CaP) scaffold embedded with the FGF2 or FGF4 variant.

Treatment Arms:

-   -   Treatment 1 (Control): 5 lambs (5 limbs), no treatment    -   Treatment 2: 5 lambs (5 limbs): CaP alone    -   Treatment 3: 5 lambs (5 limbs): CaP with FGF2 or FGF4 variant

Materials and Methods:

Lambs are assigned randomly into one of the five treatment arms.Surgical lengthening of the right femur is performed in 25 lambs agedfrom 3 to 4 months.

Anesthesia and Pre-Mediation:

General anesthesia is given without endotracheal intubation.Intramuscular atropine is given as premedication (0.5 mg/kg), andthiopentone sodium-2.5% (10-15 mg/kg), Fentanyl® (0.0015 mg/kg) andDiazepam® (0.2 mg/kg) is administered intravenously.

Fixation:

A monolateral external fixator (Monotube-Triax®, Stryker Trauma, Geneva,Switzerland) with four pins, two proximal and two distal in each of itspin clamps, is positioned so that the pins are kept away from the growthplates and the surface of the joint. The osteotomy is performed using apneumatic saw.

Lengthening:

Lengthening begins seven days after surgery for all treatment groups:Lengthening continues until the limb has been lengthened by 4.5 cm. Thetotal elongation period lasts 30 days at a rate of 1.5 mm/day startingthe 8 h day after surgery.

Treatment:

Lambs are assigned randomly into one of the four treatment arms. Alltreatments take place during the consolidation period, at day 44.

Treatment 1 (control): To assess the effect during the consolidationperiod, animals remain without treatment until the end of the trialperiod.

Treatment 2: To assess the effect of CaP alone during the consolidationperiod, it will be administered once, one week after completion ofelongation.

Treatment 3: To assess the effect of the variant protein duringconsolidation period, CaP with FGF2 or FGF4 variant is administeredonce, one week after completion of elongation.

Follow Up:

Animals are in a restricted area during the extent of the wholeexperiment and are allowed to feed and walk ad libitum in own cage.Animals are weighed at fixed intervals and general well-being ismonitored.

To study the bone formation in the host bone, four differentfluorochromes are used as bone markers, administered 1M, according tothe following schedule: one week after surgery: calcein (green; Sigma®);two weeks after surgery: alizarin (red; Sigma®); three weeks aftersurgery: xylenol (orange; Fluka®) and three days before sacrificeoxytetracycline will be given (Duphacycline®). The Spalteholz techniqueis performed after intra-arterial injection of Berlin blue studiedthrough the femoral artery before sacrifice to analyze thevascularization of the lengthened callus in each group.

Completion:

The animals are sacrificed three months after initial surgery by IVinjection of 5 μg of KCl, after anesthesia with sodium pentobarbital(1.5 mg/kg weight).

Assessment of Efficacy:

Success is determined in terms of healing time and bone quality obtainedafter elongation and treatment with FGF2 or FGF4 variant and if no majoradverse effects are observed.

X-ray:

Progress of bone healing is followed by X-ray at weeks 1, 2 and 4 afterbeginning of elongation. The parameters to be assessed from the X-rayare:

-   -   1. Degree of callus formation    -   2. Gap closure    -   3. Remodeling achieved during treatment.

X-ray scoring is performed by an orthopedic surgeon, according to anestablished bone healing grading system, according to the following: Nocallus-0, primary callus response at one end of bone-1, primary cluusresponse at both ends of bone-2, partial external callus union-3,complete external callus union-4, <30% gap closure-5, >30% gapclosure-6, complete gap closure-7, partial callus remodeling-8, completecallus remodeling-9; Gap filling calculation: (A/A+B)×100 equals thepercentage of gap filling.

Histology:

The callus is divided into two parts, one for embedding in paraffin, andthe other for embedding in methylmethacrylate. For the histologicalstudy, the specimens will be fixed in Bouin for 24 hours and decalcifiedin a solution of PVP-EDTA, at 4° C. Once specimens have beendecalcified, they are dehydrated using increasing concentration ofalcohols (70%, 80%, 96% and 100%), and after 4 hours in xylene, they areembedded in paraffin at a temperature of 60° C. The specimens aresectioned to 4 μm, and stained with Masson's trichrome, hematoxylin andeosin (H&E), Safranin O and von Kossa.

To analyze the mineralization by fluorochromes, the specimens are fixedin formol for one week, then dehydrated using alcohols of increasingproof. After one week in PMMA-alcohol and three weeks in PMMA (Technovit7200 VLC®), specimens will be sectioned with a diamond saw (Exakt®) andtrimmed to a thickness of 14 μm. After measuring the sections withultraviolet light the distance of the bone markers is measured and thebone index formation calculated (distance mm/days). The proximal partsof both, lengthened and control, tibiae are extracted and cut in lateraland medial parts. The lateral portion is placed in 4% bufferedformaldehyde. After decalcification of all the specimens in EDTA, areproceed to embed them in paraffin and cut them into 4 μm slices. Stainsof H&E, Masson's trichrome, Safranin O and Alcian blue-PAS are applied.

Immunohistochemistry:

Specific antibodies recognizing collagen L collagen II, FGFa (FGF1), andS-100 are applied to the lengthened callus sections by an indirecttwo-step method The 4 μm paraffin sections are dewaxed in xylene andtaken through ethanol 100%. After trypsinization, followingdeparaffinization, endogenous peroxidase is blocked by placing thesections in hydrogen peroxidase solution for 30 min. They are thenincubated in the following reagents with appropriateTris-buffered-saline (TBS: 0.55 M, pH 7.36) washes: normal pig serum for30 min, abovementioned primary antibodies for 1 hour, a secondarybiotinylated antibody for 30 min, and avidin-biotin complex (Dako KO355)for 30 min. The reaction is visualized with chromogen substrate solution(diaminobenzidine, hydrogen peroxidase, TB) and sections arecounterstained with Harris's hematoxylin, dehydrated, and mounted. As anegative control, TBS is used in the procedure instead of the primaryantibodies. All stained sections are examined and photographed with useof a microscope (Nikon Optiphot-2®, Japan).

Morphometric Analysis:

With an image analyzing system (Leica Q 500 MC®) the histomorphometricparameters are determined. With Masson's trichrome stain the followingparameters are determined:

-   -   1. Trabecular width.    -   2. Trabecular area.    -   3. Trabecular erosion surface.    -   4. Index of trabecular erosion.    -   5. Number of osteoblasts.    -   6. Number of osteoclasts per field.    -   7. Number of osteoclast nuclei.    -   8. Index of bone reabsorption or number osteoclast        nuclei/osteoclasts.

With von Kossa's stain the following parameters are obtained.

-   -   1. Osteoid width.    -   2. Osteoid-trabecular index, and fluorescence will be used to        measure.    -   3. Bone formation index.

Example 17 Goat Articular Cartilage Repair Model

A comparative study to evaluate the efficacy of the FGF variants intreating articular cartilage defects in a goat knee injury model isperformed. A total of 6 adult female goats are used. AR of the animalsundergo a chondrocyte harvest procedure prior to implantation. Thecollected tissue will be used for preparation of autologous primarychondrocytes. Three weeks post operative, a 4.5 mm diameter and 1.5 mmdeep hole are punched out and natural matrix matrices, with or withoutFGF variants, pre-seeded with different concentrations of allogeneiccells are implanted in the corresponding individual goat for a long termexperiment (12 weeks). After 12 weeks, all animals are humanelyeuthanized. The joints are grossly evaluated for specific changes of thefemoral condyle and the contacting surfaces. Histological analysis isperformed to determine the structural and cellular response to theimplant materials.

Materials and Methods:

Six adult female goats (11-12 months old) are used. In one particularexperimental system the different tests include:

Antibiotics: 2 ml of amoxycillin is injected IM immediately before theprocedure and once a day for 4 days after the procedure.

Anesthesia: Pre-medication: 0.05 mg/kg xylazine followed byketamine-diazepam (4 mg/kg and 2 mg/kg IV) is administered IM.

Surgery and Implantation:

The basic surgical procedure is identical for all subjects. Allsurgeries are performed under strict asepsis. Peri-operative antibioticsare dosed IM at 2.4 million units of Penicillin procaine (40,000units/kg SID) at the beginning of the procedure. Anesthesia is inducedwith xylazine 0.05 mg/kg IM followed by ketamine-diazepam (4 mg/kg and 2mg/kg IV). The subject is intubated in ventral position and thenpositioned to left recumbency. Anesthesia is maintained with a gaseousmixture of Isoflurane and oxygen. Analgesia, carprofen 2-4 mg/kg SQ,SID.

Harvest Procedure:

The surgical approach consists of a curved, lateral skin incision madefrom the distal one-third of the left femur to the level of the tibialplateau and across to the medial side of the tibial spine. Using thismethod, the skin is bluntly dissected and retracted to allow a lateralparapatellar approach into the stifle joint. An incision is madeparallel to the lateral border of the patella and patellar ligament.This extends from the lateral side of the fascia lata along the cranialborder of the biceps femoris and into the lateral fascia of the stiflejoint. The biceps femoris and attached lateral fascia are retractedallowing an incision into the joint capsule. The joint is extended andthe patella luxated medially exposing the stifle joint.

The harvest site is the same as the location of the planned trochleardefect which is created in the right femoral condyle. The defects isapproximately 5 mm in diameter and approximately 2.5 mm in depth, andwill pass into the subchondral bone. The defects are made on either thelateral or medial wall of the distal trochlear sulcus dependent onindividual anatomy. The harvested cartilage layer is approximately 5 mmin diameter and approximately 0.5 mm in depth. The harvested cells aretransferred to cell culture medium immediately after harvest forexpansion and matrix seeding. The incision is closed in layers usingappropriate suture and patterns.

Implantation Procedure:

The trochlear defect is created in the right femoral condyle. Thedefects are approximately 5 mm in diameter and approximately 2.5 mm indepth, and pass into the subchondral bone. The defects are made oneither the lateral or medial wall of the distal trochlear sulcusdependent on individual anatomy. Each defect is filled with theappropriate test article.

The surgical approach consists of a curved, lateral skin incision madefrom the distal one-third of the left femur to the level of the tibialplateau and across to the medial side of the tibial spine. Using thismethod, the skin is bluntly dissected and retracted to allow a lateralparapatellar approach into the stifle joint. An incision is madeparallel to the lateral border of the patella and patellar ligament.This extends from the lateral side of the fascia lata along the cranialborder of the biceps femoris and into the lateral fascia of the stiflejoint. The biceps femoris and attached lateral fascia are retractedallowing exposure to the joint capsule. The joint is extended and thepatella luxated medially exposing the stifle joint.

With the knee joint fully flexed, the appropriate location for thepoints of drilling the defect on the trochlear sulcus are identified andmarked with a surgical marker. A specially designed cartilage cutter isused to slice through the cartilage outer layer and prevent tearing ofthe cartilage. The approximate 5 mm diameter core cutter is used underpower to create a fixed depth of approximately 2.5 mm, maintaining aplane perpendicular to the tangent of the sulcus. The core ofsubchondral bone and cartilage is carefully removed. The cutter iscarefully removed and any loose cartilage edging is carefully dissectedwith a scalpel blade. If needed, a handheld powered drill with aspecially designed drill bit is used to chamfer the edge of the createddefect. This undercutting may assist in providing a mechanical lock withthe matrix.

The cartilage defects are copiously flushed with sterile saline prior toinsertion of the test article. The appropriate test material is thenplaced into the defect such that it is in line with the surroundingcartilage and covered with biological glue to maintain in place. A finalsaline flush of the joint is carefully done. The patella is then reducedand the joint moved through a complete range-of-motion to ensure thatthere is no impingement due to the implants. This is followed by routineclosure of the joint in three or four layers using appropriate suturematerial.

Post operatively, a modified Thomas splint is applied to the leg. Thisremains in place for 2 weeks to limit flexing of the operated knee. Postoperative checks are made for any animal displaying signs of postoperative discomfort. Post operative analgesics are given for 5 days ifthe animals display any signs of distress of discomfort. All treatmentsare recorded in the appropriate study documentation.

Necropsy:

Animals are humanely sacrificed at 12 weeks postoperatively. Bodyweightsare recorded immediately prior to sacrifice. Deep anesthesia is inducedwith a mixture of ketamine-xylazine and the subject exsanguinatedaccording to the guidelines set forth by the AVMA Panel on Euthanasia(JAVMA, March 2000).

Gross evaluation and sample collection are performed. Lymph node inclose proximity to the joint is examined. The articulating surfacesopposing the defect sites are examined for any abnormal joint surface.Additionally, gross evaluations of the knee joints are made to determinethe cartilage repair. Femora, patellae, synovium, and popliteal lymphnodes are harvested and placed into appropriately labeled containers.Immediately following tissue harvest, gross morphological examination ofthe cartilage surface is done as described above and photographicrecords made of each specimen.

Gross Morphological Observations:

After collection of the knee joints, the joints are opened, photographedand the surface of the defect site is scored. The synovial membrane isexamined for any inflammation. Joint fluid is collected and analyzed.

Histology and Histological Evaluation:

Immediately after dissection and following gross joint surfaceobservations, the joints is placed in 10% phosphate buffered formalin(at least ten-fold volume) for at least 48 hours and sent forhistological processing. After fixation in 10% phosphate bufferedformalin, the specimens are grossly trimmed to remove extra tissue. Thetissue blocks are cut approximately ⅓ of the distance in from theexterior implant/tissue interface in order to examine them grossly.Contact radiographs are taken prior to the commencement ofdecalcification.

The tissues are decalcified in 10% EDTA until radiographs of thedecalcified sections assures complete decalcification. Once completedecalcification is determined, the specimens are dehydrated through anethanol series and paraffin embedded. The specimens are sectioned. Onesection is stained with H&E and another sequential section with SafraninO counter-stained with Fast Green. For histologic analysis of thesections, the scoring scale according to Frenkel is used.

Histological evaluation is performed to measure the followingparameters: Characteristics of the neo-formed tissue, regularity of thejoint surface of the regenerated tissue, structural integrity andthickness of the regenerated tissue, endochondral ossification and stateof the cells in the remaining cartilage.

Example 18 Pharmacokinetics

Methods for detecting administered compounds in the blood or tissue oftreated mammals are known in the art. The pharmacokinetic properties ofthe administered compounds are determined using such methods. In animalmodels, radiolabelled oligonucleotides or peptides can be administeredand their distribution within body fluids and tissues assessed byextraction of the oligonucleotides or peptides followed byautoradiography. Other methods include labeling of a peptide with areporter moiety, including fluorescent or enzyme labels, administrationto an animal, extraction of the peptide from body fluids and organsfollowed by HPLC analysis. Alternatively, immunohistochemical methodsare used for detection of the administered peptide in tissue. Thepresent invention contemplates reporter labeled FGF polypeptides andchimeras, fusion protein, hybrids and conjugates using the same.

Results:

FGF4: Activity and specificity of hFGF4^(Δ78) and FGF4^(Δ54N165R)

The hFGF4^(Δ78) N-terminal polypeptide was disclosed in Bellosta et al.(2001) as an FGF4 ligand having wild type characteristics. TheN-terminal truncated FGF4^(Δ54) and the N-terminal truncated and mutatedFGF4^(Δ54N165R), which were disclosed in International patentapplication WO 03/094835 were shown to have normal activity towardsFGFR3 and FGFR1 and thus were used for comparison.

FDCP-FGFR1, FGFR2 or FGFR3 cells were grown with increasing FGF4^(Δ78)or FGF4^(Δ54N165R) concentrations and the resulting proliferationresponse was measured by XTT. FDCP-FGFR1 and FDCP-FGFR2 cells respondedsimilarly to both ligands. Unexpectedly, the FGF4^(Δ78) behaved as apoor ligand for FGFR3, inducing a growth response at only 25 ng/ml andmore, while FDCP-FGFR3 cells proliferate at as little as 0.1 ng/mlhFGF4^(Δ54N165R).

To precisely map the amino-terminal domain required for full activity ofFGF4, a series of one amino acid successive N-terminal deletions,Δ72-Δ77, was prepared. These constructs were generated by PCR, clonedinto p89, expressed and purified as described for the above variants.FDCP-FGFR1 and FGFR3 cells were exposed to the different truncatedmutants for 2 days and then followed cell proliferation. FIG. 2 showshFGF4^(Δ72) and hFGF4^(Δ73) induced FDCP-FGFR3 cell proliferation aswell as the wild-type FGF4, yet larger deletions demonstrated decreasingactivation of the FGFR3 bearing cells. Such reduced activity was notobserved with FDCP-FGFR1 cells, emphasizing the importance of aminoacids 74-78 for FGFR3 activation.

FGF2: Activity and Specificity of N-Truncated FGF2 Polypeptides

A series of FGF2 N-terminal truncated variants were prepared. TheFGF2^(Δ26 variant was tested in FDCP proliferation assays. FGFR)2 andFGFR3 activation by FGF2^(Δ26) is severely reduced while that of FGFR1remained intact as compared to the activation by the wild-type FGF2ligand (F2-WT; FIG. 3A-3C).

The ligands were then tested in the RCJ system. RCJ cells expressingeither FGFR1, FGFR2 or FGFR3 were stimulated with an increasing dose ofwild-type or mutant FGF2 and lysates from stimulated cells were analyzedby Western with a phospho MAPK (antiphospho ERK; FIG. 4). The activityof FGF2^(Δ26) mirrored that observed in the FDCP cell system showingequal activation, as the wild-type, of MAPK in RCJ-FGFR1 cells and arelatively reduced one in RCJ-FGFR2 and -FGFR3 cells. Thus the increasedspecificity of N-terminal truncated FGF2 might be relevant to a broadrange of biological systems. FIGS. 5A and 5B show the proliferativeresponse of FGFR1 (A) and FGFR3 (B) expressing cells to FGF2^(Δ26) andFGF2³¹ variant ligands.

In an effort to map the N-terminal residues required for FGFR2, FGFR3and FGFR4 recognition successively smaller N-terminal variants that lackeither residues 2-25 (FGF2^(Δ25)) or 2-24 (FGF2^(Δ24)) were generatedand subjected to a proliferation assay of FDCP-FGFR cells. BothFGF2^(Δ25) and FGF2^(Δ24) demonstrated potent activation of FGFR1similar to that obtained with FGF2^(Δ26) (FIG. 6A). As demonstratedpreviously, FGF2^(Δ26) was a poor activator of the other 3 receptors(the activity towards FGFR2 is shown in FIG. 8M). The activity ofFGF2^(Δ26N111G) was similar to that of the FGF2^(Δ26) variant. It did,however, show a larger stability in comparison to FGF2^(Δ26) lacking theN111G mutation. The addition of a single amino acid to the FGF2^(Δ26),namely phenylalanine 25 in FGF2^(Δ25) reactivated, at least in part, itsability to induce a proliferative response in cells expressing FGFR3 andFGFR4. In line with these data, FGF2^(Δ24) demonstrated wild-typeactivity in FDCP-FGFR3 and FDCP-FGFR4 cells suggesting thatphenylalanine 24 and histidine 25 may be required for full activation ofthese receptors (FIG. 6B-6C).

The nearly full activation of FGFRs by FGF2^(Δ24) compared to theremarkable FGFR1 specificity of FGF2^(Δ26) suggests that positions 25and 26 are important for receptor binding. A set of 15 oligonucleotidesthat encompass these 2 positions and code for representatives of acidic,basic, polar or non-polar amino acids were designed at these two sites.The 15 mutants were cloned in p89 as Δ24 N-terminal truncated ligands.Production of 14 out of 15 mutants was demonstrated (Δ24H25QF26Qexpression was undetectable). The mutants were purified usingheparin-Sepharose® beads and quantified by gel densitometry.

The mutant ligands were then added at increasing dose to FDCP-FGFR1cells and the resulting cell proliferation was measured by XTT analysis(FIGS. 7A-7N; Table V).

TABLE V Activation of FGFR1-4 by N-terminal truncated FGF2 variants SEQID NO: FGFR1 FGFR2 FGFR3 FGFR4 FGF2 (wt) 3 ++++ ++++ ++++ ++++FGF2^(Δ24) 5 ++++ +++ +++ +++ FGF2^(Δ25) 6 ++++ +++ ++ ++ FGF2^(Δ26) 7++++ ++ + + Δ24H25D 17 ++++ +++ +++ ++ Δ24H25Q 18 ++++ +++ +++ ++Δ24H25V 19 ++++ +++ +++ + Δ24H25K 20 ++++ +++ + + Δ24F26D 21 +++ + − −Δ24F26Q 22 ++++ + − + Δ24F26K 23 +++ + − + Δ24F26Y 24 ++++ +++ +++ +++Δ24H25DF26D 25 ++++ ++ + − Δ24H25DF26Q 26 ++++ ++ − − Δ24H25DF26K 27++++ + − − Δ24H25QF26D 28 ++++ ++ + − Δ24H25QF26K 29 ++++ + − −Δ24H25VF26D 30 ++++ ++ − + Δ24H25VF26Q 31 ++++ ++ + + Δ24H25VF26K 32++++ + − − Δ24H25KF26Q 33 +++ − − −

All mutants activated FGFR1 efficiently yet slight differences wereobserved (Table V). Specifically, all single mutants with substitutionat His25 were slightly more active towards FGFR1 than wild-type FGF2while those harboring a mutation at Phe26, either as a single or doublemutant were as active as, or slightly less active than the wild-type.These mutants were then analyzed in FDCP cells that express the otherthree receptor types.

This experiment showed that the single His mutants, FGF2^(Δ24H25D),FGF2^(Δ24H25Q) and FGF2^(Δ24H25V) at least as active as FGF2^(Δ24) inactivating FGFR2 (FIG. 8; Table V). In contrast, all variants withsubstitution at Phe26 were less active than FGF2^(Δ24) and exceptFGF2^(Δ24H25DF26Q) and FGF2^(Δ24H25VF26D) were also less active thanFGF2^(Δ26). This suggests that the deleterious effect of mutations atPhe26 to Asp or Gln may be alleviated by the substitution of thepositively charged His at position 25 for a non-polar or a negativelycharged residue. In general, the effect of the different mutations onFGFR3 activation was very similar to that on FGFR2 except thatFGF2^(Δ24H25DF26Q) and FGF2^(Δ24H25VF26D) were as inactive as the othervariants (FIG. 9).

The only receptor that responded less to the FGF variantsFGF2^(Δ24H25D), FGF2^(Δ24H25Q), FGF2^(Δ24H25V) than to FGF2^(Δ24) wasFGFR4 (FIG. 10). This suggests that His 25 has a role in FGFR4activation and that Phe26, is important for FGFR4 activation as it isfor FGFR2 and FGFR3.

The heparin requirement of the different mutants for activating FGFR1was analyzed under very low heparin concentrations (FIG. 11). Thisallowed the discrimination between different His 25 mutants, showingthat FGF2^(Δ24H25D) requires the lowest heparin concentrations for itsbiological activity (FIG. 11A). As before, any mutation at Phe26 reducedthe ability of the ligand to induce FDCP-FGFR1 cell proliferation andall tested ligands with a double mutation at His 25 and Phe 26 requiredless heparin than the respective single Phe 26 mutants (FIG. 11B-11D).

Since a positive charge at position 25 showed a negative effect onligand activity, His 25 was replaced with amino acids that possesshigher pK values. The following mutants: FGF2^(Δ24H25K) andFGF2^(Δ24H25KF26Q) were thus prepared as described for the abovevariants and compared with FGF2^(Δ24), FGF2^(Δ24H25D), FGF2^(Δ24F26Q)and FGF2^(Δ24H25DF26Q) (FIG. 12). FDCP-FGFR3 cells (FIG. 12B) showedthat FGF2^(Δ24H25D was more potent while FGF)2^(Δ24H25K) was less potentthan FGF2^(Δ24). Moreover, the double mutant FGF2^(Δ24H25DF26Q) rescuedin part the loss of activity of FGF2^(Δ24F26Q) while FGF2^(Δ24H25KF26Q)was as inactive as FGF2^(Δ24F26Q). In general, FDCP-FGFR1 cells (FIG.12A) are strongly activated even by the mutant FGF variants. However,qualitative differences similar to those found in FDCP-FGFR3 cells werealso observed in FDCP-FGFR1 cells. Hence, replacing His 25 with aminoacids that have higher pK values rendered the mutant less active thanwild-type FGF2.

To further confirm the role of Phe 26 in receptor binding, a mutationwas introduced in the context of wild-type FGF2. The activity ofFGF2^(F26Q) was compared to that of wild-type FGF2. XTT analysis in thepresence of saturated heparin concentrations showed similar FGFRsactivation by both ligands (FIGS. 13A-13C). However, when the activityof the FGF2^(F26Q) mutant was compared to that of the wild-type FGF2under limiting concentrations of heparin, the mutant ligand wasconsiderably less active than the wild-type FGF2 in FDCP-FGFR2 andFDCP-FGFR3 (FIGS. 13E-13F) but not in FDCP-FGFR1 cells (FIG. 13D).

In contrast to the non-conservative mutations described above, aconservative mutation at position 26 replacing Phe26 with Tyr(FGF2^(Δ24F26Y)) did not alter the activity of the mutant compared toits native counterpart (FGF^(Δ24)), (FIG. 14; Table V). Thus, aromaticamino acids at position 26 fulfill the structural requirements for FGFRactivation.

FGF2 and FGF4 Variants: Chondrogenic and Osteogenic Potential

The chondrogenic and osteogenic potential of bone marrow stem cells wasdetermined by culturing the cells in FGF2 or FGF4 variants of thepresent invention. The cells cultured in the presence of the differentligands were examined for their in-vitro chondrogenic potential usingthe pellet culture assay. The effect of novel FGF2 variants, namelyFGF2^(Δ26) and FGF2^(Δ31) on primary human articular chondrocytesproliferation is shown in FIG. 15. Both FGF2 variants enhanced theproliferation rate of primary human articular chondrocytes.

A high proliferation rate was observed when both the FGF2 variants(FGF2^(Δ24F26Q) and FGF2^(Δ26)) and the FGF4 variant (FGF4^(Δ78)) wereincluded in the medium of all bone marrow samples tested (FIG. 16). Theproliferation rate of the FGF2 variants was very similar and higher thanwith no ligand. The proliferation rate of FGF4^(Δ78) was slightly lowerthan that of the FGF2 variants, yet it showed a higher proliferationrate in comparison to the one lacking a ligand. The chondrogenicpotential of the chondrocytes cultured with the FGF2 and FGF4 variantswas improved and showed stronger marker for hyaline cartilage.

Results of pellet cultures histology of the bone marrow stem cellsshowed that cells cultured with FGF2 and FGF2^(Δ26) gave pellet culturesimilar in size and with massive amount of GAG's and sulfated GAG's(FIG. 17). It seems that cells cultured with FGF2 showed slightly largersignal in Safranin O stain (SO) than cells cultured with FGF2^(Δ26).Also, high amount of Collagen II was observed by immunohistochemistrywith anti Col II antibodies of the pellets produced from the cellscultured with FGF2 and FGF2^(Δ26) variant. Human bone marrow derivedmesenchymal stem cells expanded without FGF showed poor chondrogenicpotential in comparison to that obtained with FGF2 variants FGF2^(Δ24),FGF2^(Δ26) and FGF2^(Δ31) (FIG. 18).

qPCR analysis of Col II in pellet culture produced from human bonemarrow derived mesenchymal stem cells expanded with different FGF2variants of the present invention, showed high chondrogenic potential(FIG. 19). Moreover, the chondrogenic potential of hBM-MSC cultured withFGF2^(Δ31) was higher than those of cells cultured with FGF2^(Δ26).FGF2^(Δ26) showed similar results to FGF2^(Δ24F26Q) yet significantlyhigher chondrogenic potential than FGF2. In general the qPCR results,which provide RNA information, are in good agreement with thechondrogenic potential observed for the FGF variants.

To examine the multipotency of the expanded bone marrow stem cells withthe various ligands an in-vitro osteogenic assay was set up. Resultsshown in FIG. 20 showed very similar intensity of Alizarin red stain forall cultures.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1.-45. (canceled)
 46. An isolated variant of a fibroblast growth factor(FGF) polypeptide selected from the group consisting of FGF2 and FGF4comprising an N-terminal deletion; wherein the variant polypeptideretains between 0 and 11 amino acid residues at the N-terminus extendingbeyond the Leu-Tyr-Cys (LYC) motif of the β1 strand of the core domain;and wherein the variant polypeptide has increased receptor selectivitywhen compared to the corresponding isolated wild type FGF polypeptide bya gain of activity or loss of activity by at least a factor of twotoward at least one receptor subtype but not toward all fibroblastgrowth factor receptor (FGFR) subtypes.
 47. The isolated FGF variantpolypeptide according to claim 46, wherein the FGF is FGF2, and whereinthe isolated FGF2 variant polypeptide retains between 0 and 5 amino acidresidues at the N-terminus extending beyond the LYC motif of the β1strand of the core domain.
 48. The isolated FGF variant polypeptideaccording to claim 47, having a sequence as set forth in any one of SEQID NOS: 7-12.
 49. The isolated FGF variant polypeptide according toclaim 46, wherein the FGF is FGF2 and wherein the isolated FGF2 variantpolypeptide further comprises at least one additional modification inits polypeptide sequence, the modification selected from an amino aciddeletion, an amino acid substitution and an amino acid insertion. 50.The isolated FGF variant polypeptide according to claim 49, furthercomprising at least one amino acid substitution in the beta8-beta9 loop,wherein the polypeptide has a sequence set forth in SEQ ID NO:94. 51.The isolated FGF variant polypeptide according to claim 50, having asequence set forth in SEQ ID NO:13.
 52. The isolated FGF variantpolypeptide according to claim 49, further comprising at least one aminoacid substitution in the retained N-terminus.
 53. The isolated FGFvariant polypeptide according to claim 52, having a sequence as setforth in any one of SEQ ID NOS:14-16, 17-23 or 25-33.
 54. The isolatedFGF variant polypeptide according to claim 46, wherein the FGF is FGF4and wherein the isolated FGF4 variant polypeptide retains between 8 to11 amino acid residues at the N-terminus extending beyond the LYC motifof the β1 strand of the core domain.
 55. The isolated FGF variantpolypeptide according to claim 54, having a sequence as set forth in anyone of SEQ ID NOS: 63-65.
 56. A polynucleotide sequence encoding anisolated variant of a fibroblast growth factor polypeptide according toclaim
 46. 57. The polynucleotide sequence according to claim 56, as setforth in any one of SEQ ID NOS: 36-42, 43-49, 51-59, or 72-74.
 58. Avector comprising a polynucleotide sequence according to claim
 56. 59. Ahost cell comprising a vector according to claim
 58. 60. Apharmaceutical composition comprising as an active ingredient at leastone isolated variant of a fibroblast growth factor polypeptide accordingto claim 46, or a polynucleotide sequence encoding the isolated FGFpolypeptide variant; and a pharmaceutically acceptable diluent orcarrier.
 61. The pharmaceutical composition according to claim 60formulated for administration via intra-articular, intravenous,intramuscular, subcutaneous, intradermal, or intrathecal routes.
 62. Thepharmaceutical composition according to claim 60 formulated foradministration to the site of a bone fracture or for wound healing orfor treatment of coronary or peripheral vascular disease.
 63. A methodof treating an individual having a skeletal disorder or a coronary andperipheral vascular disease comprising the step of administering to thatindividual a pharmaceutical composition according to claim
 60. 64. Themethod according to claim 63, wherein the treatment of a skeletaldisorder is designated in promoting or enhancing bone fracture healingor growth processes.
 65. The method according to claim 63, wherein thepharmaceutical composition comprises an isolated FGF variant having apolypeptide sequence as set forth in SEQ ID NO:
 66. 66. A method ofinducing cellular expansion, comprising the steps of: isolating apopulation of cells to be expanded; and exposing said cells to anN-terminal truncated polypeptide variant according to claim
 46. 67. Themethod of claim 66, wherein the population of cells to be expandedcomprises cells selected from hematopoietic cells, stem cells orprogenitor cells.
 68. The method of claim 66, wherein the population ofcells to be expanded comprises cells selected from chondrocytes,osteoblasts, hepatocytes, fibroblasts or mesenchymal, endothelial,epithelial, urothelial, endocrine, neuronal, pancreatic, renal or ocularcell types.
 69. An FGF2 variant having a sequence as set forth in SEQ IDNO:90 or
 91. 70. A pharmaceutical composition comprising as an activeingredient the FGF2 variant according to claim 69; and apharmaceutically acceptable diluent or carrier.